Inhibitors of ncca-atp channels for therapy

ABSTRACT

Methods and compositions are provided that are utilized for treatment and/or prevention of intraventricular hemorrhage or progressive hemorrhagic necrosis (PHN), particularly following spinal cord injury. In particular, the methods and compositions are inhibitors of a particular NC Ca-ATP  channel and include, for example, inhibitors of SUR1 and/or inhibitors of TRPM4. Kits for treatment and/or prevention of intraventricular hemorrhage or progressive hemorrhagic necrosis (PHN), particularly following spinal cord injury, are also provided. The present invention also concerns treatment and/or prevention of intraventricular hemorrhage in infants, including premature infants utilizing one or more inhibitors of the channel is provided to the infant, for example to brain cells of the infant.

This application is a continuation of U.S. patent application Ser. No.14/040,104 filed Sep. 27, 2013; which is a continuation of U.S. Pat. No.8,557,867 granted Oct. 15, 2013, formerly application Ser. No.12/665,853 filed Jan. 21, 2010; which is a U.S. §371 national phaseapplication of International Patent Application No. PCT/US2008/067640filed Jun. 20, 2008; which claims priority to U.S. Provisional PatentApplication No. 60/945,825 filed Jun. 22, 2007; and to U.S. ProvisionalPatent Application No. 60/945,811 filed Jun. 22, 2007; and to U.S.Provisional Patent Application No. 60/945,636 filed Jun. 22, 2007, allof which applications are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant NumbersNS048260 and HL082517 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention concerns at least the fields of cell biology,molecular biology, and medicine. In particular aspects, the presentinvention concerns the fields of treatment and/or prevention ofintraventricular hemorrhage or spinal cord injury, particularly relatedto progressive hemorrhagic necrosis, for example.

BACKGROUND OF THE INVENTION

The present invention concerns therapy for a variety of maladies,including at least spinal cord injury and intraventricular hemorrhage.

Spinal Cord Injury

Acute spinal cord injury (SCI) results in physical disruption of spinalcord neurons and axons leading to deficits in motor, sensory, andautonomic function. SCI is a debilitating neurological disorder commonin young adults that often requires life-long therapy and rehabilitativecare, placing significant burdens on healthcare systems. Although manypatients exhibit neuropathologically and clinically complete cordinjuries following SCI, many others have neuropathologically incompletelesions (Hayes and Kakulas, 1997; Tator and Fehlinds, 1991) giving hopethat proper treatment to minimize “secondary injury” may reduce thefunctional impact.

The concept of secondary injury in SCI arises from the observation thatthe lesion expands and evolves over time (Tator and Fehlings, 1991; Kwonet al., 2004). Whereas primary injured tissues are irrevocably damagedat the time of impact, tissues that are destined to become “secondarily”injured are considered to be potentially salvageable. Older observationsbased on histological studies that gave rise to the concept oflesion-evolution have been confirmed with non-invasive MRI (Bilgen etal., 2000).

Several mechanisms of secondary injury have been postulated, includingischemia/hypoxia, oxidative stress and inflammation, all of which havebeen considered to be responsible for the devastating process termed“progressive hemorrhagic necrosis” (PHN) (Tator and Fehlings, 1991;Nelson et al., 1977; Tator, 1991; Fitch et al., 1999; Tator andKoyanagi, 1997). PHN is a mysterious condition, first recognized overthree decades ago, that has thus far eluded understanding and treatment.Shortly after injury (10-15 min), a small hemorrhagic lesion involvingprimarily the capillary-rich central gray matter is observed, but overthe following 3-24 h, petechial hemorrhages emerge in more distanttissues, eventually coalescing into the characteristic lesion ofhemorrhagic necrosis (Balentine, 1978; Kawata et al., 1993). The whitematter surrounding the hemorrhagic gray matter shows a variety ofabnormalities, including decreased hematoxylin and eosin staining,disrupted myelin, and axonal and periaxonal swelling. White matterlesions extend far from the injury site, especially in the posteriorcolumns (Tator and Koyanagi, 1997). The evolution of hemorrhage andnecrosis has been referred to as “autodestruction”. PHN results in lossof vital spinal cord tissue and, in some species including humans, leadsto post-traumatic cystic cavitation surrounded by glial scar tissue.

The mechanism responsible for PHN is not known. Tator and Koyanagi(1997) speculated that obstruction of small intramedullary vessels bythe initial mechanical stress or secondary injury might be responsiblefor PHN, whereas Kawata and colleagues (Kawata et al., 1993) attributedthe progressive changes to leukocyte infiltration around the injuredarea leading to plugging of capillaries. Given that petechialhemorrhages, the pathognomonic feature of PHN, form as a result ofcatastrophic failure of vascular integrity, damage to the endothelium ofspinal cord capillaries and postcapillary venules has long been regardedas a major factor in the pathogenesis of PHN (Nelson et al., 1977;Griffiths et al., 1978; Kapadia, 1984). However, no molecular mechanismfor progressive dysfunction of endothelium has been identified.

The sulfonylurea receptor-1 (SUR1)-regulated NC_(Ca-ATP) channel is anon-selective cation channel that is not constitutively expressed, butis transcriptionally up-regulated in astrocytes and neurons following anhypoxic or ischemic insult (Chen and Simard, 2001; Chen et al., 2003;Simard et al., 2006). The channel is inactive when expressed, butbecomes activated when intracellular ATP is depleted, with activationleading to cell depolarization, cytotoxic edema and oncotic cell death.Block of the channel in vitro by the sulfonylurea, glibenclamide,prevents cell depolarization, cytotoxic edema and oncotic cell deathinduced by ATP depletion. In rodent models of ischemic stroke, treatmentwith glibenclamide results in significant improvements in edema, lesionvolume and mortality (Simard et al., 2006). In humans with diabetesmellitus, use of sulfonylureas before and during hospitalization forstroke is associated with significantly better stroke outcomes (Kunte etal., 2007).

Intra-Axial Hemorrhage

Intra-axial hemorrhage is characterized by bleeding within the brainitself. Intraparenchymal or intraventricular hemorrhages are types ofintra-axial hemorrhage.

Intraventricular Hemorrhage (IVH)

Intraventricular Hemorrhage (IVH), a bleeding from fragile blood vesselsin the brain, is a significant cause of morbidity and mortality inpremature infants and may have include, for example, death,shunt-dependent hydrocephalus, and life-long neurological consequencessuch as cerebral palsy, seizures, mental retardation, and otherneurodevelopmental disabilities. Neurological sequelae includeshunt-dependent hydrocephalus, seizures, neurodevelopmentaldisabilities, and cerebral palsy. The vasculature is especially fragilein preterm infants, particularly those born more than 8 weeks early,i.e., before 32 weeks of gestation. IVH is more commonly seen inextremely premature infants; its incidence is over 50% in preterminfants with birth weight less than 750 grams, and up to 25% in infantswith birth weight less than 1000 to 1500 grams.

IVH encompasses a wide spectrum of intra-cranial vascular injuries withbleeding into the brain ventricles, a pair of C-shaped reservoirs,located in each half of the brain near its center, that containcerebrospinal fluid. Bleeding occur in the subependymal germinal matrix,a region of the developing brain located in close proximity to theventricles. Within the germinal matrix, during fetal development, thereis intense neuronal proliferation as neuroblasts divide and migrate intothe cerebral parenchyma. This migration is about complete by about the24th week of gestation, although glial cells can still be found withinthe germinal matrix until term. The germinal matrix undergoes rapidinvolution from the 26th to the 32nd week of gestation, at which timeregression is nearly complete, as glial precursors migrate out topopulate the cerebral hemispheres.

Supporting this intense cell differentiation and proliferation activitythere is a primitive and fragile capillary network. These vessels havethin walls for their relatively large size, lack a muscularis layer,have immature interendothelial junctions and basal laminae, and oftenlack direct contact with perivascular glial structures, suggestingdiminished extravascular support. It is in this fragile capillarynetwork where IVH originates. When a fetus is born prematurely, theinfant is suddenly thrust from a well-controlled, protective environmentinto a stimulating, hostile one. Because of this physiologic stress andshock, the infant may lose the ability to regulate cerebral blood flowand may suffer alterations in blood flow and pressure and in the amountsof substances dissolved in the blood such as oxygen, glucose and sodium.The fragile capillaries may, and often do, rupture.

The severity of the condition depends on the extent of the vascularinjury. There are four grades, or stages, of IVH as can be seen usingultrasound or brain computer tomography. Grade I IVH, the less severestage, involves bleeding in the subependymal germinal matrix, with lessthan 10% involvement of the adjacent ventricles. Grade II IVH resultswhen 10 to 40% of the ventricles are filled with blood, but withoutenlargement of the ventricles. Grade III IVH involves filling of over50% of the ventricles with blood, with significant ventricularenlargement. In Grade IV IVH, the bleeding extends beyond theintraventricular area into the brain parenchyma (intraparenchymalhemorrhage).

The major complications of IVH relate to the destruction of the cerebralparenchyma and the development of posthemorrhagic hydrocephalus.Following parenchymal hemorrhages (Grade IV IVH), necrotic areas mayform cysts that can become contiguous with the ventricles. Cerebralpalsy is the primary neurological disorder observed in those cases,although mental retardation and seizures may also occur. In addition,infants affected with Grade III to IV IVH may develop posthemorrhagichydrocephalus, a condition characterized by rapid growth of the lateralventricles and excessive head growth within two weeks of the hemorrhage.Likely causes are obstruction of the cerebrospinal fluid conduits byblood clots or debris, impaired absorption of the cerebrospinal fluid atthe arachnoid villi, or both. Another form of the hydrocephaluscondition may develop weeks after the injury. In this case the likelycause is obstruction of the cerebrospinal fluid flow due to anobliterative arachnoiditis in the posterior fossa.

Several trials were conducted in the 1980s and 1990s to evaluateprophylactic use of phenobarbitone in preterm infants to reduce the riskof IVH, however, no statistical significance was observed (Postnatalphenobarbitone for the prevention of intraventricular hemorrhage inpreterm infants, Whitelaw et al., 2000; and Bedard M P, Shankaran S,Slovis T L, Pantoja A. Dayal B. Poland R L. Effect of prophylacticphenobarbital on intraventricular hemorrhage in high-risk infants.Pediatrics 1984; 73:435-9.). Other pharmacological interventions havebeen assessed, such as indomethacin (Fowlie 1999), but withoutsubstantial clinical impact and IVH remains a problem. (Whitelaw A,Placzek M, Dubowitz L, Lary S, Levene M. Phenobarbitone for preventionof periventricular haemorrhage in very low birth-weight infants. Arandomised double-blind trial. Lancet 1983; ii: 1168-70.).

Extra-Axial Hemorrhage

Extra-axial hemorrhage is characterized by bleeding that occurs withinthe skull but outside of the brain tissue. Epidural hemorrhage, subduralhemorrhage and subarachnoid hemorrhage are types of extra-axialhemorrhage.

Subarachnoid Hemorrage (SAH)

SAH, like intraparenchymal hemorrhage, may result from trauma (physicalor physiological) or from ruptures of aneurysms or arteriovenousmalformations, or a combination thereof. SAH often indicates thepresence of blood within the subarachnoid space, blood layering/layeredinto the brain along sulci and fissures, or blood filling cisterns (suchas the suprasellar cistern because of the presence of the vessels of thecircle of Willis and their branchpoints within that space). The classicpresentation of subarachnoid hemorrhage is the sudden onset of a severeheadache. This can be a very dangerous entity, and requires emergentneurosurgical evaluation, and sometimes urgent intervention. In theUnited States, the annual incidence of nontraumatic SAH is about 6-25per 100,000. Internationally, incidences have been reported but vary to2-49 per 100,000.

Unlike ischemic stroke, in SAH the entire cortex bathed in blood is atrisk from hemotoxicity-related inflammation. Also, hemotoxicity-relatedinflammation is potentially more amenable to treatment than ischemicstroke because it develops relatively slowly, compared to rapid loss ofpenumbral tissues in ischemia. At present, treatments for edema arelimited because underlying molecular mechanis are not well understood,and treatments aimed at mechanism that have been implicated (Park etal., 2004) are not yet available. Therefore, the present inventionfulfills a long-standing need in the art by providing a treatment forSAH predicated on ameliorating (or otherwise inhibiting) post-SAHhemotoxicity-related inflammation.

The present invention provides a solution for a long-felt need in theart to treat progressive hemorrhagic necrosis following spinal cordinjury and to treat IVH, traumatic brain injury, and subarachnoidhemorrhage. for example.

SUMMARY OF THE INVENTION

The present invention is directed to systems, methods, and compositionsthat concern multiple conditions, including progressive hemorrhagicnecrosis following spinal cord injury, traumatic brain injury,subarachnoid hemorrhage, and intraventricular hemorrhage, for example.

In particular embodiments, the present invention concerns a specificchannel, the NC_(Ca-ATP) channel. The NC_(Ca-ATP) channel is a uniquenon-selective cation channel that is activated by intracellular calciumand blocked by intracellular ATP. In particular aspects, the NC_(Ca-ATP)channel of the present invention has a single-channel conductance topotassium ion (K+) between 20 and 50 pS. The NC_(Ca-ATP) channel is alsostimulated by Ca²⁺ on the cytoplasmic side of the cell membrane in aphysiological concentration range, (from about 10⁻⁸ to about 10⁻⁵ M).The NC_(Ca-ATP) channel is also inhibited by cytoplasmic ATP in aphysiological concentration range (from about 0.1 mM to about 10 mM, ormore particularly about 0.2 mM to about 5 mM). The NC_(Ca-ATP) channelis also permeable at least to the following cations; K⁺, Cs⁺, Li⁺, Na⁺;with the permeability ratio between any two of the cations typicallybeing greater than about 0.5 and less than about 2, for example.

The NC_(Ca-ATP) channel includes at least a pore-forming component(pore-forming subunit) and a regulatory component (regulatory subunit);the regulatory subunit includes sulfonylurea type 1 receptor (SUR1) andthe pore-forming subunit includes a non-selective cation channel subunitthat is, or closely resembles, a transient receptor potential melastatin4 (TRPM4) pore. In some embodiments, pathological diseases andconditions may be treated or prevented by inhibition of the NC_(Ca-ATP)channel. The NC_(Ca-ATP) channel may be inhibited by reducing itsactivity, by reducing the numbers of such channels present in cellmembranes, and by other means. For example, the NC_(Ca-ATP) channel maybe inhibited by administration of SUR1 antagonists; by administration ofTRPM4 antagonists; by administration of a combination of drugs includinga SUR1 antagonist and a TRPM4 antagonist; by reducing or antagonizingthe expression, transcription, or translation of genetic messageencoding the NC_(Ca-ATP) channel; by reducing or antagonizing theinsertion of NC_(Ca-ATP) channels into cell membranes; and by othermeans.

In particular embodiments the NC_(Ca-ATP) channel is regulated bysulfonylurea receptor 1 (SUR1): e.g., it is opened by ATP depletion.SUR1-regulated NC_(Ca-ATP) channels have been shown to play an importantrole in cytotoxic edema, oncotic cell death, and hemorrhagic conversionin ischemic stroke and CNS trauma. Moreover, SUR1 is blocked by SUR1antagonists such as, for example, glibenclamide and tolbutamide,providing an exemplary avenue for treatment. TRPM4 pores may be blockedby TRPM4 antagonists (e.g., TRPM4 blockers such as, for example,pinkolant, rimonabant, or a fenamate). In one aspect, thehypoxic-ischemic environment in prematurity leads to transcriptionalactivation of SUR1 and opening of NC_(Ca-ATP) channels, initiating acascade of events culminating in acute hemorrhage in parallel withischemic stroke. Thus, hypoxic, ischemic, or hemorrhagic injury may betreated by inhibition of the NC_(Ca-ATP) channel, e.g., byadministration of a SUR1 antagonist, a TRPM4 antagonist, or both.

In certain embodiments related at least to spinal cord injury, forexample, the channel is expressed in neural, glial, and vascular cellsand tissues, among others, including in capillary endothelium, cells inthe core near the spinal cord injury impact site, and in reactiveastrocytes although in alternative cases the channel is expressed inneurons, glia and neural endothelial cells after brain trauma, forexample.

More particularly, the present invention relates to the regulationand/or modulation of this NC_(Ca-ATP) channel and how its modulation canbe used to prevent, ameliorate, or treat intraventricular hemorrhageand/or spinal cord injury and/or progressive hemorrhagic necrosis and/ortraumatic brain injury and/or subarachnoid hemorrhage or other hypoxicor ischemic injury, disease, or condition. Administration of anantagonist or inhibitor of the NC_(Ca-ATP) channel is effective tomodulate and/or regulate the channel and to prevent or treat suchinjury, disease, or condition in specific embodiments. Thus, dependingupon the disease, a composition (an antagonist, which may also bereferred to as an inhibitor) is administered to block or inhibit atleast in part the channel, for example to prevent cell death and/or toprevent or reduce or modulate depolarization of the cells.Administration of an antagonist or inhibitor of the NC_(Ca-ATP) channelincludes administration of a SUR1 antagonist, a TRPM4 antagonist, orboth, and may include such administration in combination withadministration of other agents as well.

The invention encompasses antagonists of the NC_(Ca-ATP) channel,including small molecules, large molecules, proteins, (includingantibodies), as well as nucleotide sequences that can be used to inhibitNC_(Ca-ATP) channel gene expression (e.g., antisense and ribozymemolecules). In certain cases, an antagonist of the NC_(Ca-ATP) channelincludes one or more compounds capable of one or more of the following:(1) blocking the channel; (2) preventing channel opening; (3) inhibitingthe channel; (4) reducing the magnitude of membrane current through thechannel; (5) inhibiting transcriptional expression of the channel;and/or (6) inhibiting post-translational assembly and/or trafficking ofchannel subunits.

Another aspect of the present invention for the treatment of ischemic,hypoxic, or other injury, including IVH or spinal cord injury orprogressive hemorrhagic conversion comprises administration of aneffective amount of a SUR1 antagonist and/or a TRPM4 antagonist andadministration of glucose. Glucose administration may be by intravenous,or intraperitoneal, or other suitable route and means of delivery.Additional glucose allows administration of higher doses of anantagonist of the NC_(Ca-ATP) channel than might otherwise be possible,so that combined glucose with an antagonist of the NC_(Ca-ATP) channelprovides greater protection, and may allow treatment at later times,than with an antagonist of the NC_(Ca-ATP) channel alone. Greateramounts of glucose are administered where larger doses of an antagonistof the NC_(Ca-ATP) channel are administered.

In certain aspects, antagonists of one or more proteins that comprisethe channel and/or antagonists for proteins that modulate activity ofthe channel are utilized in methods and compositions of the invention.The channel is expressed on neuronal cells, neuroglia cells, neuralepithelial cells, neural endothelial cells, vascular cells, or acombination thereof, for example. In specific embodiments, an inhibitorof the channel directly or indireclty inhibits the channel, for exampleby the influx of cations, such as Na+, into the cells, therebypreventing depolarization of the cells. Inhibition of the influx of Na+into the cells thereby at least prevents or reduces cytotoxic edemaand/or ionic edema, and/or vasogenic edema and prevents or reduceshemorrhagic conversion. Thus, this treatment reduces cell death ornecrotic death of at least neuronal, glial, vascular, endothelial,and/or neural endothelial cells.

The NC_(Ca-ATP) channel can be inhibited by an NC_(Ca-ATP) channelinhibitor, an NC_(Ca-ATP) channel blocker, a type 1 sulfonylureareceptor (SUR1) antagonist, SUR1 inhibitor, a TRPM4 inhibitor, or acompound capable of reducing the magnitude of membrane current throughthe channel, or a combination or mixture thereof. In further specificembodiments, the SUR1 inhibitor is a sulfonylurea compound or abenzamido derivative. A SUR1 inhibitor such as iptakalim may be used.More specifically, the exemplary SUR1 antagonist may be selected fromthe group consisting of glibenclamide, tolbutamide, repaglinide,nateglinide, meglitinide, midaglizole, LY397364, LY389382, glyclazide,glimepiride, estrogen, estrogen related-compounds (estradiol, estrone,estriol, genistein, non-steroidal estrogen (e.g., diethystilbestrol),phytoestrogen (e.g., coumestrol), zearalenone, etc.), and compoundsknown to inhibit or block K_(ATP) channels. MgADP can also be used toinhibit the channel. Other compounds that can be used to block orinhibit K_(ATP) channels include, but are not limited to tolbutamide,glyburide (1[p-2[5-chloro-O-anisamido)ethyl] phenyl]sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide (1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido)ethyl] phenyl] sulfonyl] urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl). In a specific embodiment, the cation channelblocker is selected from the group consisting of pinkolant, rimonabant,a fenamate (such as flufenamic acid, mefenamic acid, meclofenamic acid,or niflumic acid),1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazolehydrochloride, and a biologically active derivative thereof. Inadditional embodiments, non-sulfonyl urea compounds, such as 2,3-butanedione and 5-hydroxydecanoic acid, quinine, and therapeuticallyequivalent salts and derivatives thereof, may be employed in theinvention. The benzamido derivative may be selected from the groupconsisting of repaglinide, nateglinide, and meglitinide. The inhibitormay comprise a protein, a peptide, a nucleic acid (such as an RNAimolecule or antisense RNA, including siRNA), or a small molecule. Inspecific aspects, the inhibitor is provided intravenously,subcutaneously, intramuscularly, intracutaneously, intragastrically, ororally. In an additional embodiment, the method further comprisesadministering MgADP to the individual.

In one embodiment of the invention, NC_(Ca-ATP) channels are involved inprogressive hemorrhagic necrosis (PHN) in SCI. Although endothelialdysfunction has been implicated in PHN, SUR1-regulated NC_(Ca-ATP)channels have not previously been shown in capillary endothelium. Here,development of the present invention utilized a rodent model ofunilateral cervical SCI and endothelial cell cultures, wherein SUR1 wasprominently up-regulated in capillaries in the region of SCI,endothelial cells subjected to hypoxic conditions express SUR1-regulatedNC_(Ca-ATP) channels, and inhibition of SUR1 by a variety of molecularlydistinct mechanisms largely eliminated the progressive extravasation ofblood characteristic of PHN, reduced lesion size, and was associatedwith marked neurobehavioral functional improvement, consistent with acritical role for SUR1-regulated NC_(Ca-ATP) channels in PHN followingSCI.

Thus, in one embodiment of the invention, there is a method of treatingand/or preventing progressive hemorrhagic necrosis in an individual,comprising the step of providing to the individual an effective amountof an inhibitor of a NC_(Ca-ATP) channel. In a specific embodiment, theprogressive hemorrhagic necrosis is a direct or indirect result ofspinal cord injury. In another specific embodiment, the inhibitor of thechannel is a SUR1 inhibitor, a TRPM4 inhibitor, or a combination ormixture thereof. The inhibitor may be provided intravenously,subcutaneously, intramuscularly, intracutaneously, intragastrically, ororally. In an additional specific embodiment, the method furthercomprises administering MgADP to the individual.

An individual provided the methods of the invention may be an individualthat suffers from a spinal cord injury or that is at risk for having aspinal cord injury, for example. Individuals at risk for having spinalcord injuries may be of any kind, and in certain cases the spinal cordinjury is the result of an unexpected accident. Still, some groups ofthe population have a higher risk of sustaining a spinal cord injury,including at least, for example, men; African-Americans; young adults;seniors; motor vehicle accident victims; fall victims; victims ofviolence, for example, gunshot wounds, stabbings and assaults; athletes,including those who partake in football, rugby, wrestling, gymnastics,diving, surfing, swimming, ice hockey, equestrian activities, ordownhill skiing, for example; individuals participating in recreationalactivities, such as horseback riding, swimming; and individuals withpredisposing conditions, such as conditions that affect the bones orjoints, including arthritis or osteoporosis.

The present invention is also directed to a system and method thatconcern treatment and/or prevention of intraventricular hemorrhage in anindividual, and, in specific embodiments, in a premature infant. Inparticular aspects, a premature infant is defined as any infant that isrecognized in the art to be premature, although in specific aspects apremature infant is an infant that is born before the 37th week ofpregnancy.

The present invention relates to a novel ion channel whose functionunderlies the swelling of a cell, for example, such as in response toATP depletion. Treatment methods are provided that exploit thedifferential expression of such channels in response to trauma,including but not limited to the use of inhibitors of the channelfunction to prevent the cell swelling response. Several adverse effectsare associated with such physiological phenomenon, including hemorrhagicstroke, intracranial hemorrhage, and further, IVH and SAH.

In certain embodiments, the invention is drawn to methods of treatingintracranial hemorrhage, including but not limited to intra-axialhemorrhage such as IVH and extra-axial hemorrhage such as SAH. Inspecific embodiments, the methods comprise the administration of aninhibitor of an NC_(Ca-ATP) channel to a cell and/or subject in needthereof.

In an exemplary embodiment of the present invention, the treatmentmethods are effective for therapeutic and/or preventative compositionsand methods of the invention may be provided to the premature infantfollowing birth, the mother of the premature infant during pregnancy, orthe infant in utero. In a specific embodiment, the inhibitor is providedto the mother prior to 37 weeks of gestation. In another specificembodiment, the mother is at risk for premature labor. In a furtherspecific embodiment, the pregnancy is less than 37 weeks in gestationand the mother has one or more symptoms of labor.

Thus, in one non-limiting embodiment, there is a method of treatingintraventricular hemorrhage in the brain of an infant or preventingintraventricular hemorrhage in the brain of an infant at risk fordeveloping intraventricular hemorrhage, comprising administering aneffective amount of an inhibitor of NC_(Ca-ATP) channel to the infantfollowing birth and/or the mother prior to birth of the infant. In aspecific embodiment, the infant is a premature infant. In furtherspecific embodiments, the infant weighs less than 1500 grams at birth orweighs less than 1000 grams at birth. In particular aspects, the infantis a premature infant born at 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, or at or prior to 23 weeks of gestation.

In an additional embodiment, there is a kit for treating and/orpreventing intraventricular hemorrhage or spinal cord injury (includingrelated to PHN), comprising an inhibitor of NC_(Ca-ATP) channel,including an inhibitor of TRPM4 and/or SUR1. The channel inhibitor is aSUR1 inhibitor, a TRPM4 inhibitor, or a mixture or combination thereof,in specific embodiments. The kit may further comprise an additionalcompound for treating spinal cord. The kit may further comprise anadditional compound for treating intraventricular hemorrhage, either fordelivery to the infant and/or to the mother. In some embodiments of thekit, the kit comprises methylprednisone, one or more of a cation channelblocker, and/or an antagonist of VEGF, MMP, NOS, or thrombin, forexample. The kit may also comprise suitable tools to administercompositions of the invention to an individual. The inhibitor isformulated for administration in utero, in specific embodiments forintraventricular hemorrhage.

In yet another exemplary embodiment of the present invention, thecompositions and methods of the present invention are predicated on theconcept that cortical dysfunction is due to hemotoxcity-relatedinflammation, which activates an immune response cascade events, such asproduction of cytokines such as TNFalpha and/or NF-kappaB, resulting inupregulation of SUR1-regulated NC_(Ca-ATP) channels, therebypredisposing the cell/subject to edema and/or cell death. Thus, in anon-limiting embodiment, the invention includes methods of treatingand/or preventing SAH comprising administration of an effective amountof an inhibitor of an NC_(Ca-ATP) channel to a cell and/or subject inneed thereof.

In specific embodiments, the methods of treating or preventing SAH areuseful in any subject at risk for SAH, such as hypertensive patients,individuals at risk to trauma both physical and physiological, and thelike.

The methods of the present invention may include combination therapies,such as co-administration of dexamethasone, glucose, an antiinflammatoryagent, an anticholesterol agent, an antihyperlipoproteinemic agent, orother agent or combination of agents, for example. In certainembodiments, methods of the present invention may include combinationtherapies including antithrombotic and or antifibrinolytic agents, suchas co-administration of tPA, for example to help remove a blood clotfrom the ventricle or any condition that would not be contra-indicatedfor co-administration of tPA. In fact, one of skill in the artrecognizes that at least some of the conditions that are treatable withthe methods of the present invention (intraventricular hemorrhage,subarachnoid hemorrhage, progressive secondary hemorrhage andprogressive hemorrhagic necrosis, for example) are all situations withexcess bleeding, and tPA, anti-platelet agents and anticoagulants arecontraindicated, because they could worsen the bleeding. Such compoundswould not be utilized in cases where there is bleeding or where bleedingis suspected.

The present invention provides compounds that inhibit the NC_(Ca-ATP)channel for the treatment and/or prevention of intraventricularhemorrhage in an individual, wherein the individual is provided one ormore inhibitors of the channel. The inhibitor(s) may be of any kind, butin specific embodiments it is an inhibitor of a regulatory subunit ofthe channel and/or a pore-forming subunit of the channel. In certainaspects a combination or mixture of an antagonist of a regulatorysubunit of the channel and an antagonist of a pore-forming subunit ofthe channel are provided to the individual.

The therapeutic and/or preventative compositions and methods of theinvention may be provided to the premature infant following birth, themother of the premature infant during pregnancy, or the infant in utero.In a specific embodiment, the inhibitor is provided to the mother priorto 37 weeks of gestation. In another specific embodiment, the mother isat risk for premature labor. In a further specific embodiment, thepregnancy is less than 37 weeks in gestation and the mother has one ormore symptoms of labor.

Thus, in one embodiment, there is a method of treating intraventricularhemorrhage in the brain of an infant or preventing intraventricularhemorrhage in the brain of an infant at risk for developingintraventricular hemorrhage, comprising administering an effectiveamount of an inhibitor of NC_(Ca-ATP) channel to the infant followingbirth and/or the mother prior to birth. In a specific embodiment, theinfant is a premature infant. In further specific embodiments, theinfant weighs less than 1500 grams at birth or weighs less than 1000grams at birth. In particular aspects, the infant was born at 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, or at or prior to 23 weeksof gestation.

In one aspect, the present invention provides novel methods of treatinga patient comprising administering at least a therapeutic compound thattargets the NC_(Ca-ATP) channel, either alone or in combination with anadditional therapeutic compound, and in specific embodiments theadditional therapeutic compound is methylprednisolone, cation channelblockers and antagonists of VEGF, MMP, NOS, and/or thrombin, forexample.

In one embodiment, the therapeutic combinatorial composition can beadministered to and/or into the spinal cord injury site, for example.Such administration to the site includes injection directly into thesite, for example, particularly in the case where the site has beenrendered accessible to injection due to trauma to the spine, forexample.

Any compound(s) of the invention can be administered alimentarily (e.g.,orally, buccally, rectally or sublingually); parenterally (e.g.,intravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneously, intraperitoneally, intraventricularly);by intracavity; intravesically; intrapleurally; and/or topically (e.g.,transdermally), mucosally, or by direct injection into the brainparenchyma.

In further embodiments, the compound that inhibits the NC_(Ca-ATP)channel can be administered in combination with, for example, statins,diuretics, vasodilators (e.g., nitroglycerin), mannitol, diazoxideand/or similar compounds that ameliorate ischemic conditions. Yetfurther, another embodiment of the present invention comprises apharmaceutical composition comprising statins, diuretics, vasodilators,mannitol, diazoxide or similar compounds that ameliorate ischemicconditions or a pharmaceutically acceptable salt thereof and a compoundthat inhibits a NC_(Ca-ATP) channel or a pharmaceutically acceptablesalt thereof. This pharmaceutical composition can be consideredneuroprotective, in specific embodiments. In only certain embodiments ofthe invention, there are methods and compounds (including pharmaceuticalconditions) that concern administration in combination with a compoundthat inhibits the NC_(Ca-ATP) channel, such as a thrombolytic agent(e.g., tissue plasminogen activator (tPA), urokinase, prourokinase,streptokinase, anistreplase, reteplase, tenecteplase), an anticoagulantor antiplatelet (e.g., aspirin, warfarin or coumadin) may be employed,wherein such compounds would not be contra-indicated. For example, thepharmaceutical composition comprising a combination of the thrombolyticagent and a compound that inhibits a NC_(Ca-ATP) channel is therapeutic,because it increases the therapeutic window for the administration ofthe thrombolytic agent by several hours; for example, the therapeuticwindow for administration of thrombolytic agents may be increased byseveral hours (e.g. about 4-about 8 hrs) by co-administering one or moreantagonists of the NC_(Ca-ATP) channel.

An effective amount of an antagonist of the NC_(Ca-ATP) channel orrelated-compounds thereof as treatment and/or prevention variesdepending upon the host treated and the particular mode ofadministration. In one embodiment of the invention, the dose range ofthe therapeutic combinatorial composition of the invention, including anantagonist of NC_(Ca-ATP) channel and/or the additional therapeuticcompound, will be about 0.01 μg/kg body weight to about 20,000 μg/kgbody weight. The term “body weight” is applicable when an animal isbeing treated. When isolated cells are being treated, “body weight” asused herein should read to mean “total cell body weight”. The term“total body weight” may be used to apply to both isolated cell andanimal treatment. All concentrations and treatment levels are expressedas “body weight” or simply “kg” in this application are also consideredto cover the analogous “total cell body weight” and “total body weight”concentrations. However, those of skill will recognize the utility of avariety of dosage range, for example, 0.01 μg/kg body weight to 20,000μg/kg body weight, 0.02 μg/kg body weight to 15,000 μg/kg body weight,0.03 μg/kg body weight to 10,000 μg/kg body weight, 0.04 μg/kg bodyweight to 5,000 μg/kg body weight, 0.05 μg/kg body weight to 2,500 μg/kgbody weight, 0.06 μg/kg body weight to 1,000 μg/kg body weight, 0.07μg/kg body weight to 500 μg/kg body weight, 0.08 μg/kg body weight to400 μg/kg body weight, 0.09 μg/kg body weight to 200 μg/kg body weightor 0.1 μg/kg body weight to 100 μg/kg body weight. Further, those ofskill will recognize that a variety of different dosage levels will beof use, for example, 0.0001 μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004μg/kg, 0.005 μg/kg, 0.0007 μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg,150 μg/kg, 160 μg/kg, 180 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg,500 μg/kg, 550 μg/kg, 600 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 900μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or30 mg/kg.

An effective amount of an inhibitor of NC_(Ca-ATP) channel that may beadministered to an individual or a cell in a tissue or organ thereofincludes a dose of about 0.0001 nM to about 2000 μM, for example. Morespecifically, doses of an antagonist to be administered are from about0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM; about 0.05 μMto about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM to about 2.0μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0 μM; about4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about 10 μM toabout 50 μM; about 50 μM to about 100 μM; about 100 μM to about 200 μM;about 200 μM to about 300 μM; about 300 μM to about 500 μM; about 500 μMto about 1000 μM; about 1000 μM to about 1500 μM and about 1500 μM toabout 2000 μM, for example. Of course, all of these amounts areexemplary, and any amount in-between these dosages is also expected tobe of use in the invention.

An effective amount of an inhibitor of the NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention the dose range of the agonist or antagonist of theNC_(Ca-ATP) channel or related-compounds thereof will be about 0.01μg/kg body weight to about 20,000 μg/kg body weight.

In specific embodiments, the dosage is less than 0.8 mg/kg. Inparticular aspects, the dosage range may be from 0.005 mg/kg to 0.8mg/kg body weight, 0.006 mg/kg to 0.8 mg/kg body weight, 0.075 mg/kg to0.8 mg/kg body weight, 0.08 mg/kg to 0.8 mg/kg body weight, 0.09 mg/kgto 0.8 mg/kg body weight, 0.005 mg/kg to 0.75 mg/kg body weight, 0.005mg/kg to 0.7 mg/kg body weight, 0.005 mg/kg to 0.65 mg/kg body weight,0.005 mg/kg to 0.5 mg/kg body weight, 0.09 mg/kg to 0.8 mg/kg bodyweight, 0.1 mg/kg to 0.75 mg/kg body weight, 0.1 mg/kg to 0.70 mg/kgbody weight, 0.1 mg/kg to 0.65 mg/kg body weight, 0.1 mg/kg to 0.6 mg/kgbody weight, 0.1 mg/kg to 0.55 mg/kg body weight, 0.1 mg/kg to 0.5 mg/kgbody weight, 0.1 mg/kg to 0.45 mg/kg body weight, 0.1 mg/kg to 0.4 mg/kgbody weight, 0.1 mg/kg to 0.35 mg/kg body weight, 0.1 mg/kg to 0.3 mg/kgbody weight, 0.1 mg/kg to 0.25 mg/kg body weight, 0.1 mg/kg to 0.2 mg/kgbody weight, or 0.1 mg/kg to 0.15 mg/kg body weight, for example.

In specific embodiments, the dosage range may be from 0.2 mg/kg to 0.8mg/kg body weight, 0.2 mg/kg to 0.75 mg/kg body weight, 0.2 mg/kg to0.70 mg/kg body weight, 0.2 mg/kg to 0.65 mg/kg body weight, 0.2 mg/kgto 0.6 mg/kg body weight, 0.2 mg/kg to 0.55 mg/kg body weight, 0.2 mg/kgto 0.5 mg/kg body weight, 0.2 mg/kg to 0.45 mg/kg body weight, 0.2 mg/kgto 0.4 mg/kg body weight, 0.2 mg/kg to 0.35 mg/kg body weight, 0.2 mg/kgto 0.3 mg/kg body weight, or 0.2 mg/kg to 0.25 mg/kg body weight, forexample.

In further specific embodiments, the dosage range may be from 0.3 mg/kgto 0.8 mg/kg body weight, 0.3 mg/kg to 0.75 mg/kg body weight, 0.3 mg/kgto 0.70 mg/kg body weight, 0.3 mg/kg to 0.65 mg/kg body weight, 0.3mg/kg to 0.6 mg/kg body weight, 0.3 mg/kg to 0.55 mg/kg body weight, 0.3mg/kg to 0.5 mg/kg body weight, 0.3 mg/kg to 0.45 mg/kg body weight, 0.3mg/kg to 0.4 mg/kg body weight, or 0.3 mg/kg to 0.35 mg/kg body weight,for example.

In specific embodiments, the dosage range may be from 0.4 mg/kg to 0.8mg/kg body weight, 0.4 mg/kg to 0.75 mg/kg body weight, 0.4 mg/kg to0.70 mg/kg body weight, 0.4 mg/kg to 0.65 mg/kg body weight, 0.4 mg/kgto 0.6 mg/kg body weight, 0.4 mg/kg to 0.55 mg/kg body weight, 0.4 mg/kgto 0.5 mg/kg body weight, or 0.4 mg/kg to 0.45 mg/kg body weight, forexample.

In specific embodiments, the dosage range may be from 0.5 mg/kg to 0.8mg/kg body weight, 0.5 mg/kg to 0.75 mg/kg body weight, 0.5 mg/kg to0.70 mg/kg body weight, 0.5 mg/kg to 0.65 mg/kg body weight, 0.5 mg/kgto 0.6 mg/kg body weight, or 0.5 mg/kg to 0.55 mg/kg body weight, forexample. In specific embodiments, the dosage range may be from 0.6 mg/kgto 0.8 mg/kg body weight, 0.6 mg/kg to 0.75 mg/kg body weight, 0.6 mg/kgto 0.70 mg/kg body weight, or 0.6 mg/kg to 0.65 mg/kg body weight, forexample. In specific embodiments, the dosage range may be from 0.7 mg/kgto 0.8 mg/kg body weight or 0.7 mg/kg to 0.75 mg/kg body weight, forexample. In specific embodiments the dose range may be from 0.001 mg/dayto 3.5 mg/day. In other embodiments, the dose range may be from 0.001mg/day to 10 mg/day. In other embodiments, the dose range may be from0.001 mg/day to 20 mg/day.

Further, those of skill will recognize that a variety of differentdosage levels will be of use, for example, 0.0001 μg/kg, 0.0002 μg/kg,0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007 μg/kg, 0.001 μg/kg, 0.1μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0 μg/kg, 10.0 μg/kg, 15.0μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg, 90 μg/kg, 100 μg/kg,120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180 μg/kg, 200 μg/kg, 225μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg,400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 700 μg/kg, 750μg/kg, 800 μg/kg, 900 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 12 mg/kg, 15mg/kg, 20 mg/kg, and/or 30 mg/kg. In particular embodiments, there maybe dosing of from very low ranges (e.g. 1 mg/kg/day or less; 5 mg/kgbolus; or 1 mg/kg/day) to moderate doses (e.g. 2 mg bolus, 15 mg/day) tohigh doses (e.g. 5 mg bolus, 30-40 mg/day; and even higher). Of course,all of these dosages are exemplary, and any dosage in-between thesedosages is also expected to be of use in the invention. Any of the abovedosage ranges or dosage levels may be employed for an agonist orantagonist, or both, of NC_(Ca-ATP) channel or related-compoundsthereof.

An effective amount of a therapeutic composition of the invention,including an antagonist of NC_(Ca-ATP) channel and/or the additionaltherapeutic compound, that may be administered to a cell includes a doseof about 0.0001 nM to about 2000 μM, for example. More specifically,doses to be administered are from about 0.01 nM to about 2000 μM; about0.01 μM to about 0.05 □μM; about 0.05 μM to about 1.0 μM; about 1.0 μMto about 1.5 μM; about 1.5 μM to about 2.0 μM; about 2.0 □μM to about3.0 μM; about 3.0 μM to about 4.0 μM; about 4.0 μM to about 5.0 μM;about 5.0 μM to about 10 μM; about 10 μM to about 50 μM; about 50 μM toabout 100 μM; about 100 μM to about 200 μM; about 200 μM to about 300μM; about 300 □μM to about 500 μM; about 500 μM to about 1000 μM; about1000 μM to about 1500 μM and about 1500 μM to about 2000 μM, forexample. Of course, all of these amounts are exemplary, and any amountin-between these dosages is also expected to be of use in the invention.

In particular embodiments, there may be dosing of from very low ranges(e.g. for glyburide 1 mg/day or less) to moderate doses (e.g. 3.5mg/day) to high doses (e.g. 10-40 mg/day; and even higher). Of course,all of these dosages are exemplary, and any dosage in-between thesedosages is also expected to be of use in the invention. Any of the abovedosage ranges or dosage levels may be employed for an agonist orantagonist, or both, of NC_(Ca-ATP) channel or related-compoundsthereof.

In a particular embodiment, the dosage is about 0.5 mg/day too about 10mg/day.

In certain embodiments, the amount of the combinatorial therapeuticcomposition administered to the subject is in the range of about 0.0001μg/kg/day to about 20 mg/kg/day, about 0.01 μg/kg/day to about 100μg/kg/day, or about 100 μg/kg/day to about 20 mg/kg/day. Still further,the combinatorial therapeutic composition may be administered to thesubject in the form of a treatment in which the treatment may comprisethe amount of the combinatorial therapeutic composition or the dose ofthe combinatorial therapeutic composition that is administered per day(1, 2, 3, 4, etc.), week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5,etc.), etc. Treatments may be administered such that the amount ofcombinatorial therapeutic composition administered to the subject is inthe range of about 0.0001 μg/kg/treatment to about 20 mg/kg/treatment,about 0.01 μg/kg/treatment to about 100 μg/kg/treatment, or about 100μg/kg/treatment to about 20 mg/kg/treatment.

A typical dosing regime consists of a loading dose designed to reach atarget agent plasma level followed by an infusion of up to 7 days tomaintain that target level. One skilled in the art will recognize thatthe pharmacokinetics of each agent will determine the relationshipbetween the load dose and infusion rate for a targeted agent plasmalevel. In one example, for intravenous glyburide administration, a 15.7μg bolus (also called a loading dose) is followed by a maintenance doseof 0.3 μg/min (432 μg/day) for 120 hours (5 days). This dose regime ispredicted to result in a steady-state plasma concentration of 4.07ng/mL. In another example for intravenous glyburide, a 117 μg bolus doseis followed by a maintenance dose of 2.1 μg/min (3 mg/day) for 3 days.This dose is predicted to result in a steady-state plasma concentrationof 28.3 ng/mL. In yet another example for glyburide, a 665 μg bolus doseis followed by a maintenance dose of 11.8 μg/min (17 mg/day) for 120hours (5 days). This dose is predicted to result in a steady-stateplasma concentration of 160.2 ng/mL. Once the pharmacokinetic parametersfor an agent are known, loading dose and infusion dose for any specifiedtargeted plasma level can be calculated. As an illustrative case forglyburide, the bolus is generally 30-90 times, for example 40-80 times,such as 50-60 times, the amount of the maintenance dose, and one ofskill in the art can determine such parameters for other compounds basedon the guidance herein.

In cases where combination therapies are utilized, the components of thecombination may be of any kind. In specific embodiments, the componentsare provided to an individual substantially concomitantly, whereas inother cases the components are provided at separate times. The ratio ofthe components may be determined empirically, as is routine in the art.Exemplary ratios include at least about the following: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60,1:70, 1:80, 1:90, 1:100, 1:500, 1:750, 1:1000, 1:10000, and so forth.

In particular embodiments, there may be dosing of from very low ranges(e.g. 1 mg/kg/day or less; 5 mg/kg bolus; or 1 mg/kg/day) to moderatedoses (e.g. 2 mg bolus, 15 mg/day) to high doses (e.g. 5 mg bolus, 30-40mg/day; and even higher). Of course, all of these dosages are exemplary,and any dosage between these points is also expected to be of use in theinvention. Any of the above dosage ranges or dosage levels may beemployed for an antagonist of NC_(Ca-ATP) channel or related-compoundsthereof and, in appropriate cases, of an additional compound.

In certain embodiments, the amount of the singular or combinatorialtherapeutic composition administered to the subject is in the range ofabout 0.0001 μg/kg/day to about 20 mg/kg/day, about 0.01 μg/kg/day toabout 100 μg/kg/day, or about 100 μg/kg/day to about 20 mg/kg/day. Stillfurther, the combinatorial therapeutic composition may be administeredto the subject in the form of a treatment in which the treatment maycomprise the amount of the combinatorial therapeutic composition or thedose of the combinatorial therapeutic composition that is administeredper day (1, 2, 3, 4, etc.), week (1, 2, 3, 4, 5, etc.), month (1, 2, 3,4, 5, etc.), etc. Treatments may be administered such that the amount ofcombinatorial therapeutic composition administered to the subject is inthe range of about 0.0001 μg/kg/treatment to about 20 mg/kg/treatment,about 0.01 μg/kg/treatment to about 100 μg/kg/treatment, or about 100μg/kg/treatment to about 20 mg/kg/treatment.

In those cases wherein more than one compound is provided to anindividual to treat intraventricular hemorrhage or spinal cord injuryand, in particular, progressive hemorrhagic necrosis, the compounds maybe provided in a mixture, may be provided simultaneously, or may beprovided sequentially. In cases where more than one composition isprovided to the individual, they may be provided in a particular ratioincluding, for example, in a 1:1 ratio, a 1:2 ratio, a 1:3 ratio, a 1:4ratio, and so forth.

In one embodiment of the invention, there is a composition, comprising acompound that inhibits a NC_(Ca-ATP) channel and an additionaltherapeutic compound, wherein the additional therapeutic compound isselected from the group consisting of: a) one or more cation channelblockers; and b) one or more of a compound selected from the groupconsisting of one or more antagonists of vascular endothelial growthfactor (VEGF), one or more antagonists of matrix metalloprotease (MMP),one or more antagonists of nitric oxide synthase (NOS), one or moreantagonists of thrombin, aquaporin, or a biologically active derivativethereof, wherein the NC_(Ca-ATP) channel has the followingcharacteristics: 1) it is a non-selective monovalent cation channel; 2)it is activated by an increase in intracellular calcium or by a decreasein intracellular ATP, or both; and 3) it is regulated by a SUR1.

In a further specific embodiment, one or more antagonists of vascularendothelial growth factor (VEGF) are soluble neuropilin 1 (NRP-1),undersulfated LMW glycol-split heparin, VEGF TrapR1R2, Bevacizumab,HuMV833, s-Flt-1, s-Flk-1, s-Flt-1/Flk-1, NM-3, GFB 116, or acombination or mixture thereof. In an additional specific embodiment,the undersulfated, LMW glycol-split heparin comprises ST2184. In anadditional specific embodiment, the one or more antagonists of matrixmetalloprotease (MMP) are(2R)-2-[(4-biphenylsulfonyl)amino]-3-phenylproprionic acid, GM-6001,TIMP-1, TIMP-2, RS 132908, batimastat, marimastat, a peptide inhibitorthat comprises the amino acid sequence HWGF, or a mixture or combinationthereof.

In one aspect of the invention, the one or more antagonists of nitricoxide synthase (NOS) are aminoguanidine (AG),2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT), S-ethylisothiourea(EIT), asymmetric dimethylarginine (ADMA), N-nitro-L-argininemethylester (L-NAME), nitro-L-arginine (L-NA), N-(3-aminomethyl)benzylacetamidine dihydrochloride (1400W), NG-monomethyl-L-arginine(L-NMMA), 7-nitroindazole (7-NINA), N-nitro-L-arginine (L-NNA), or amixture or combination thereof. In another aspect of the invention, theone or more antagonists of thrombin are ivalirudi, hirudin, SSR182289,antithrombin III, thrombomodulin, lepirudin, P-PACK II(d-Phenylalanyl-L-Phenylalanylarginine-chloro-methyl ketone 2 HCl),(BNas-Gly-(pAM)Phe-Pip), Argatroban, and mixtures or combinationsthereof.

In a specific embodiment wherein an additional compound other than thechannel inhibitor is employed, the compound that inhibits theNC_(Ca-ATP) channel and the additional therapeutic compound aredelivered to the individual successively. In another specificembodiment, the compound that inhibits the NC_(Ca-ATP) channel isdelivered to the individual prior to delivery of the additionaltherapeutic compound. In a further specific embodiment, the compoundthat inhibits the NC_(Ca-ATP) channel is delivered to the individualsubsequent to delivery of the additional therapeutic compound. Inanother aspect, the compound that inhibits the NC_(Ca-ATP) channel andthe additional therapeutic compound are delivered to the individualconcomitantly. In an additional aspect, the compound that inhibits theNC_(Ca-ATP) channel and the additional therapeutic compound beingdelivered as a mixture. In an additional embodiment, the compound thatinhibits the NC_(Ca-ATP) channel and the additional therapeutic compoundact synergistically in the individual. In a particular case, thecompound that inhibits the NC_(Ca-ATP) channel and/or the additionaltherapeutic compound is delivered to the individual at a certain dosageor range thereof, such as is provided in exemplary disclosure elsewhereherein.

In particular embodiments, the methods of the invention are employedwithin a certain amount of time of a spinal cord injury orintraventricular hemorrhage, for example. In specific embodiments, thecomposition(s) is delivered to the individual within minutes, hours,days, or months of the injury. In further specific embodiments, thecomposition(s) are delivered to the individual within 10 minutes, within15 minutes, within 30 minutes, within 45 minutes, within 60 minutes,within 75 minutes, within 90 minutes, within 2 hours, within 2.5 hours,within 3 hours, within 3.5 hours, within 4 hours, within 4.5 hours,within 5 hours, within 5.5 hours, within 6 hours, within 6.5 hours,within 7 hours, within 7.5 hours, within 8 hours, within 8.5 hours,within 9 hours, within 9.5 hours, within 10 hours, within 10.5 hours,within 11 hours, within 11.5 hours, within 12 hours, within 13 hours,within 14 hours, within 15 hours, within 16 hours, within 17 hours,within 18 hours, within 20 hours, within 22 hours, within 24 hours, andso on, of the time of the spinal cord injury. In specific cases, thecomposition(s) of the invention are present at places where spinal cordinjury may occur (swimming pools, stables, ski resorts, gymnasiums,nursing homes, sports arenas or fields, schools, etc.), are present infirst aid kits, are present in emergency vehicles, are present inhospitals, including emergency rooms, and/or are present in doctors'offices.

In a specific embodiment of the invention, the compound that inhibitsthe NC_(Ca-ATP) channel is glibenclamide, and the maximum dosage ofglibenclamide for the individual is about 20 mg/day. In a furtherspecific embodiment, the compound that inhibits the NC_(Ca-ATP) channelis glibenclamide, and the dosage of glibenclamide for the individual isbetween about 2.5 mg/day and about 20 mg/day. In an additional specificembodiment, the compound that inhibits the NC_(Ca-ATP) channel isglibenclamide, and the dosage of glibenclamide for the individual isbetween about 5 mg/day and about 15 mg/day. In another specificembodiment, the compound that inhibits the NC_(Ca-ATP) channel isglibenclamide, and the dosage of glibenclamide for the individual isbetween about 5 mg/day and about 10 mg/day. In a still further specificembodiment, the compound that inhibits the NC_(Ca-ATP) channel isglibenclamide, and the dosage of glibenclamide for the individual isabout 7 mg/day.

In one exemplary embodiment concerning singular therapeutic compositionsof the invention, there is a method of inhibiting neural cell swellingin an individual having traumatic brain injury, cerebral ischemia,central nervous system (CNS) damage, peripheral nervous system (PNS)damage, cerebral hypoxia, or edema, comprising delivering to theindividual a therapeutically effective amount of an antagonist of TRMP4.In specific embodiments, the antagonist of TRMP4 is a nucleic acid (suchas a TRMP4 siRNA, for example), a protein, a small molecule, or acombination thereof. In particular aspects, the method further comprisesdelivering to the individual a therapeutically effective amount of anadditional therapeutic compound selected from the group consisting of:a) a SUR1 antagonist; b) one or more cation channel blockers; b) one ormore of a compound selected from the group consisting of one or moreantagonists of vascular endothelial growth factor (VEGF), one or moreantagonists of matrix metalloprotease (MMP), one or more antagonists ofnitric oxide synthase (NOS), one or more antagonists of thrombin,aquaporin, a biologically active derivative thereof, and a combinationthereof; and d) a combination thereof.

In one embodiment of the invention, there is a method for processing aninsurance claim for treatment of a medical condition of the inventionusing a composition(s) of the invention. In a specific embodiment, themethod employs a computer for said processing. In further specificembodiments, the dosage for the composition may be any suitable dosagefor treatment of the medical condition.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIGS. 1A-1G shows that SUR1 is up-regulated in SCI. FIG. 1A:Immunohistochemical localization of SUR1 in control and at differenttimes post-SCI, as indicated, with montages constructed from multipleindividual images, and positive labeling shown in black pseudocolor.FIG. 1B: Magnified views of SUR1 immunolabeled sections taken fromcontrol and from the “core” (heavily labeled area in FIG. 1A, 6 h).FIGS. 1C, 1D: Immunolabeling of capillaries with vimentin andco-labeling with SUR1 in control (FIG. 1C), and from the “penumbra”(tissue adjacent to the heavily labeled core in FIG. 1A, 6 h) (FIG. 1D).FIG. 1E: Western blots for SUR1 of spinal cord tissue from control(lanes 1,2), 6 h post-SCI (lanes 3,4) and from an equivalent amount ofblood (BL) as is present in the injured cord (lane 5); 50 μg protein inlanes 1-4, 2 μl blood in lane 5; blots representative of 5-6 CTR and SCIrats. FIGS. 1F, 1G: In situ hybridization for SUR1 in controls and inwhole cords (FIG. 1F) or in the penumbra (FIG. 1G) 6 h post-SCI usingantisense (AS) and sense (SE), as indicated. Images ofimmunohistochemistry and in situ hybridization representative offindings in 3-5 rats/group.

FIGS. 2A-2D. SUR1-regulated NC_(Ca-ATP) channel is up-regulated inendothelial cells by hypoxia. FIG. 2A: Immunolabeling and Western blots(lanes 1,2) for SUR1 in human aortic endothelial cells (ENDO) culturedunder normoxic (N) or hypoxic (H) conditions, as indicated; Westernblots for SUR1 of rat insulinoma RIN-m5F cells (INSUL; lanes 3,4)cultured under normoxic or hypoxic condition, with β-actin also shown.FIGS. 2B, 2C: Whole-cell currents during ramp pulses (4/min; HP, −50 mV)or at the holding potential of −50 mV, before and after application ofdiazoxide (FIG. 2B) or Na azide (FIG. 2C), in endothelial cells exposedto normoxic or hypoxic conditions; the difference currents are alsoshown (red); data are representative of 7-15 recordings from humanaortic endothelial cells (FIG. 2B) or bEnd.3 cells (FIG. 2C) for eachcondition. FIG. 2D: Single channel recordings of inside-out patches withCs⁺ as the principal cation, with channel openings inhibited by ATP onthe cytoplasmic side; channel amplitude at various potentials indicateda slope conductance of 37 pS (data from 7 patches) from human brainmicrovascular endothelial cells.

FIGS. 3A-3E. Block of SUR1 reduces hemorrhage after SCI. FIG. 3A: wholecords and longitudinal sections of cords 24 h post-SCI, fromvehicle-treated (CTR) and glibenclamide-treated (GLIB) rats; whitecircles indicate impact area. FIG. 3B: Cord homogenates in test tubes at24 h, and spectrophotometric measurements of blood in cord homogenatesat various times post-SCI, from vehicle-treated (CTR; n=66) andglibenclamide-treated (GLIB; n=62) rats; *, P<0.05; **, P<0.01; ***,P<0.001. FIG. 3C: Cord sections immunolabeled for vimentin to showcapillaries, at two magnifications, from SCI rats treated with vehicle(CTR) or glibenclamide (GLIB); central canal marked by arrows; imagesrepresentative of findings in 6 rats/group. FIG. 3D: Zymography ofrecombinant MMP-2 and MMP-9 performed under control conditions (CTR), inthe presence of glibenclamide (10 μM; GLIB), and in the presence ofMMP-inhibitor II (300 nM; Calbiochem). FIG. 3E: bleeding times inuninjured rats infused with vehicle (CTR) or glibenclamide (GLIB); 3rats/group.

FIGS. 4A-4D. Block of SUR1 reduces lesion size and improvesneurobehavioral function after SCI. FIGS. 4A-4C: Cord sectionsimmunolabeled for GFAP (FIG. 4A) or stained with Eriochrome cyanine-R(FIG. 4B) or hematoxylin and eosin (FIG. 4C), 1 d (FIGS. 4A and 4B) or 7d (FIG. 4C) post-SCI, from vehicle-treated (CTR) andglibenclamide-treated (GLIB) rats; images representative of findings in3 rats/group. FIG. 4D: Cascaded outlines of lesion areas in serialsections 250 μm apart, 7 d post-SCI, from vehicle-treated (CTR) andglibenclamide-treated (GLIB) rats; lesion volumes from vehicle-treated(CTR) and glibenclamide-treated (GLIB) rats (n=4-6/group; excludes 2 CTRrats that died). FIG. 4E: Performance on inclined plane (head-up andhead-down), ipsilateral paw placement and vertical exploration(rearing), at the times indicated post-SCI, in vehicle-treated (CTR) andglibenclamide-treated (GLIB) rats (same rats as in d); paw placementmeasured 1 d post-SCI; *, P<0.05; **, P<0.01; ***, P<0.001.

FIGS. 5A-5D. Gene suppression of SUR1 blocks expression of functionalNCCa-ATP channels and improves outcome in SCI. FIG. 5A: Western blotsfor SUR1 in gliotic capsule from rats with infusion of Scr-ODN (lanes1,2) or AS-ODN (lanes 3,4) directly into the brain injury site for 10-12d prior to tissue harvest; densitometric analysis of Western blots fromthe same groups of rats (n=3/group). FIG. 5B: Membrane potential ofastrocytes from gliotic capsules of the same groups of rats, duringapplication of Na azide to deplete ATP; the average depolarization inthe 2 groups is shown; 3 cells/group. FIG. 5C: Cord sectionsimmunolabeled for SUR1, 1 d post-SCI, from rats treated with i.v.infusion of Scr-ODN or AS-ODN; quantitative immunofluorescence for thesame groups of rats; (n=3/group). FIG. 5D: measurements of blood in cordhomogenates, performance on angled plane, and vertical exploration, 1 dpost-SCI, for rats treated with i.v. infusion of Scr-ODN or AS-ODN; *,P<0.05; **, P<0.01.

FIGS. 6A-6B demonstrate a western blot validating the specificity of theanti-SUR1 antibody (FIG. 6B) compared to an anti-FLAG control (FIG. 6A).

FIGS. 7A-7H show that SUR1 is upregulated in human SCI. FIGS. 7A-7H: Lowpower (FIGS. 7A-7D) and high power (FIGS. 7E-7H) views of cord sectionsstained with H&E (FIGS. 7A, 7B, 7E-7H) or immunolabeled for SUR1;sections from the core of the lesion (FIGS. 7A, 7C, 7E, 7G) or fromuninvolved cord (FIGS. 7B, 7D, 7F, 7H).

FIGS. 8A-8D demonstrate that SUR1 is upregulated in human SCI. FIGS.8A-8D: Sections from core of the lesion immunolabeled for SUR1, showingexpression in microvessels (FIG. 8A), in ballooned neuron (FIG. 8B), andin microvessels and arterioles (FIGS. 8C, 8D).

FIG. 9 demonstrates that a knockout of SUR1 gene is associated withsignificantly better short-term neurobehavioral outcome post-SCI. Spinalcord injury was produced by impact on the right side of the dura afterlaminectomy at T9. Hindpaw function was assessed 24 hr post-SCI usingthe Basso Mouse Scale for locomotion. In WT mice, function ipsilateralto the injury was absent whereas in SUR1-KO, function was preserved. InWT mice, function contralateral to the lesion was significantly moreimpaired than in SUR1-KO mice. An important element of the unilateralinjury model is that it clearly demonstrates spread of progressivehemorrhagic necrosis and prevention of that spread by SUR1-KO.

FIGS. 10A-10D show that SUR1 is upregulated by prenatalischemia/hypoxia. FIGS. 10A-10D: Progenitor cells in periventricularzones (FIG. 10A) and veins scattered throughout the basal forebrain(FIGS. 10B-10D) showed prominent upregulation of SUR1 (red); nucleilabeled with DAPI (blue).

FIGS. 11A-11M show that SUR1 and HIF1 are upregulated in the germinalmatrix of premature infants. A-C: Low power micrographs (FIGS. 11A, 11B)or montage of micrographs (FIG. 11C) of periventricular tissue stainedwith H&E (FIG. 11A), showing densely packed neural progenitor cells ofthe GM, with an arrow pointing to a small intraparenchymal hematoma, orlabeled for mRNA for Abcc8, which encodes SUR1, using in situhybridization (FIG. 11B), or immunolabeled for SUR1 (FIG. 11C); thelatter two demonstrate regionally-specific labeling for SUR1 mRNA andprotein in the GM; the montage in (FIG. 11C) shows positiveimmunolabeling in black pseudocolor; case #9 in Table 1: prematureinfant of 22 wk gestation who lived ˜12 hr and was hypoxic prior todeath, necessitating intubation and mechanical ventilation; post-morteminterval, 3 hr. FIGS. 11D-11F: Micrographs of cortical tissues (FIG.11D) or GM tissues (FIGS. 11E, 11F) processed for in situ hybridizationfor mRNA for Abcc8, using antisense probe (FIGS. 11D, 11E) or senseprobe (FIG. 11F). FIGS. 11G-11J: Micrographs of GM tissues immunolabeledfor SUR1 (red, CY3 for SUR1, and blue, DAPI for nuclei), anddouble-labeled for von Willebrand factor (green; FIGS. 11I and 11Jonly); co-labeling is indicated by yellow color; SUR1 was identified inneural progenitor cells (FIG. 11G), and in thin-walled veins frominfants with GMH (FIG. 11H, red and FIG. 11I, yellow) but not in aninfant without GMH (FIG. 11J, green); FIGS. 11H, 11I, 11J are from cases#11, 10, 1 in Table 1, respectively. FIGS. 11K-11M: Low (FIG. 11K) andhigh (FIGS. 11L, 11M) power micrographs of sections immunolabeled forHIF1α (green, FITC for HIF1α, and blue, DAPI for nuclei), showing HIF1αin a microvessel (FIG. 11L) and in neural progenitor cells (FIG. 11M).In FIGS. 11D-11M, the bars represent 50 μm.

FIG. 12 illustrates exemplary events in the germinal matrix of prematureinfants. Scheme depicting the reciprocal relationship between O₂ tensionon the one hand, and HIF1 activation and SUR1 expression on the otherhand. Mild hypoxia, which may be the norm due to the ventriculopetalblood supply, promotes neurogenesis, whereas moderate hypoxia maypromote apoptosis resulting in involution of the GM. More severe hypoxiamay promote expression of SUR1-regulated NC_(Ca-ATP) channels, whichremain inactive until critical ATP depletion is reached (˜30 μM), atwhich point the channels open, leading to oncotic death of cells,including endothelial cells, thereby compromising the structuralintegrity of veins and predisposing to GMH during episodes of venoushypertension.

FIG. 13 shows a pressure wave produced by percussion injury model.Typical pressure wave produced by 10-cm drop of 10 gm weight to produce2.5-3.0 atm peak pressure, resulting in moderate-to severe percussioninjury.

FIGS. 14A-4B show that a percussion TBI model produces deep contusioninjury. FIGS. 14A, 14B: Unprocessed (FIG. 14A) and Niss1 stained (FIG.14B) coronal sections from two different rats 24 hr followingmoderate-to-severe percussion injury (2.5-3 atm) to the posteriorparasagittal parietal cortex, note extensive hemorrhagic contusioninvolving cortex, corpus callosum and underlying hippocampus.

FIGS. 15A-E demonstrates that SUR1 is upregulated in a rat model ofpercussion TBI. FIGS. 15A, 15B: Montages of sections immunolabeled forSUR1 3 hr (FIG. 15A) and 24 hr (FIG. 15B) post-TBI (2.5-3 atm), showingprogressive upregulation of SUR1 beyond regions of necrosis; rat in(FIG. 15B) same as in FIG. 14B. FIGS. 15C, 15D: High power views ofpenumbral tissue 24-hr post-TBI immunolabeled for SUR1 (FIG. 15C) andcolabeled for vimentin (FIG. 15D) to show capillaries. FIG. 15E: Westernblots for SUR1 for uninjured rat brain, including parietal cortex andunderlying hippocampus (Sham) and for the same regions 24 hr post-TBI;β-actin shown as loading control.

FIGS. 16A-16F demonstrate that SUR1 is upregulated in human brainfollowing gunshot wound (GSW). FIGS. 16A-16F: High power views ofneurons (FIGS. 16A-16C) and capillaries (FIGS. 16D-16F) immunolabeledfor either NeuN (FIG. 16A) or vimentin (FIG. 16D) and double labeled forSUR1 (FIGS. 16B, 16E); superimposed images are also shown (FIGS. 16C,16F); biopsy specimen from 24 year old male obtained at the time ofdecompressive craniotomy/debridement, 24 hr following GSW to the brain.

FIGS. 17A-17C show that progressive secondary hemorrhage post-TBI isreduced by glibenclamide. FIGS. 17A, 17B: Unprocessed coronal sectionsshowing contusion injury in vehicle-treated control (FIG. 17A) and inglibenclamide-treated rat (FIG. 17B) 24-hr post-TBI (2.5-3 atm). FIG.17C: Extravasated blood quantified at various times post-TBI invehicle-treated and glibenclamide-treated rats, with non-linearleastsquares fit to Boltzman equation indicating half maximum blood at5.2 hr; representative brain homogenates at 24 hr from both groups arealso shown (insert); n=3-5/group; **, P<0.01.

FIG. 18 demonstrates that glibenclamide does not inhibit matrixmetalloproteinase (MMP) activity. Zymography showed that gelatinaseactivity of recombinant MMP (Chemicon) was the same under controlconditions (CTR) and in the presence of glibenclamide (10 μM), but wassignificantly reduced by MMP-inhibitor II (300 nM; Calbiochem).

FIGS. 19A-19D demonstrate that glibenclamide reduces lesion size andspares hippocampal neurons post-TBI. FIGS. 19A-19D: Low-power (FIGS.19A, 19B) and high-power (FIGS. 19C, 19D) views of Niss1-stained coronalsections 7 days post-TBI (2.5-3 atm), with high-power views showingipsilateral hippocampus; note overall loss of neurons, with manyremaining neurons pyknotic, in vehicle-treated rat (FIG. 19C) versusnormal appearance of hippocampus in glibenclamide-treated rat (FIG.19D); note hemosiderin staining (yellow discoloration) invehicle-treated rat (FIG. 19C); percussion site marked by asterisk; datashown are representative of 5 rats/group.

FIG. 20 demonstrates that glibenclamide improves neurobehavioralfunction post-TBI. Images of rats in the cylinder used to assessspontaneous forelimb use (SFU) and spontaneous vertical exploration(SVE) post-TBI (2.5-3 atm). SVE, quantified as the time (in sec) spentwith both forepaws raised above shoulder-height during the first 3 minin the cylinder, was significantly greater in glibenclamide treated ratscompared to vehicle-treated rats during repeated sessions over the firstweek post-injury; 5 rats/group; P<0.01 by repeated measures ANOVA; samerats as in FIG. 19.

FIG. 21 shows that TRPM4 physically associates with SUR1 to form theSUR1-regulated NC_(Ca-ATP) channel. Western blot for TRPM4 of totallysate (TL) of injured tissues (middle lane), and of the product ofimmunoprecipitation using SUR1 antibody (Co-IP) (right lane); ladderalso shown (left lane) (from Simard et al., submitted).

FIGS. 22A-22C demonstrate that TRPM4 is upregulated in penumbralcapillaries 24 hr post-TBI. FIGS. 22A-22C: Low-power (FIGS. 22A, 22B)and highpower (FIG. 22C) views of uninjured control (FIG. 22A) andpost-TBI penumbral (FIGS. 22B, 22C) tissues immunolabeled for TRPM4 orvon-Willebrand factor (vWf), as indicated; merged images also shown(FIG. 22C, right panel).

FIGS. 23A-23C show patch clamp of endothelial cells attached to freshlyisolated brain capillaries. FIG. 23A: Micrograph of capillaries isolatedusing magnetic particles (black clump at top of figure); arrows point tosegments targeted for patch clamp. FIGS. 23B, 23C: Currents (FIG. 23B)and I-V curve of peak currents (FIG. 23C) recorded from endothelialcells still attached to capillary; standard physiological solutionsinside and outside; n=5.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences, for example.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of” or“consist of” one or more sequences of the invention, for example. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The present application incorporates by reference herein in theirentirety the following applications: U.S. patent application Ser. No.11/099,332, filed Apr. 5, 2005; U.S. patent application Ser. No.11/359,946, filed Feb. 22, 2006; U.S. patent application Ser. No.11/229,236, filed Sep. 16, 2005; U.S. patent application Ser. No.11/574,793, filed Jul. 25, 2005; U.S. Patent Application Ser. No.60/880,119, filed Jan. 12, 2007; and U.S. Patent Application Ser. No.60/889,065, filed Feb. 9, 2007.

I. Exemplary Definitions

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one wouldconsider equivalent to the recited value (e.g., having the same functionor result). In some instances, the term “about” may include numericalvalues that are rounded to the nearest significant figure.

As used herein, the term “antagonist” refers to a biological or chemicalagent that acts within the body to reduce the physiological activity ofanother chemical or biological substance. In the present invention, theantagonist blocks, inhibits, reduces and/or decreases the activity of aNC_(Ca-ATP) channel of any cell. In the present invention, theantagonist combines, binds, associates with a NC_(Ca-ATP) channel of acell, such as an endothelial cell, including cells in capillaryendothelium, neurons or neuron-like cells, or reactive astrocytes, forexample, such that the NC_(Ca-ATP) channel is closed (deactivated),meaning reduced biological activity with respect to the biologicalactivity in the diseased state. In certain embodiments, the antagonistcombines, binds and/or associates with a regulatory subunit of theNC_(Ca-ATP) channel, particularly a SUR1: combines, binds, and/orassociates with a pore-forming subunit of the NC_(Ca-ATP) channel, suchas TRPM4; or both. The terms antagonist or inhibitor can be usedinterchangeably.

As used herein, antagonists, inhibitors, and blockers of the NC_(Ca-ATP)channel are those agents that reduce the activity or expression of theNC_(Ca-ATP) channel, and may include (but are not limited to) SUR1antagonists, TRPM4 antagonists, anti-sense molecules that inhibitexpression of the NC_(Ca-ATP) channel, MgADP, blockers of K_(ATP)channel, agents that inhibit incorporation of the NC_(Ca-ATP) channelinto the cell membrane, and other compounds and agents that prevent orreduce the activity of the NC_(Ca-ATP) channel. For example,non-sulfonyl urea compounds, such as 2, 3-butanedione and5-hydroxydecanoic acid, quinine, and therapeutically equivalent saltsand derivatives thereof, may be employed as antagonists, inhibitors, andblockers of the NC_(Ca-ATP) channel. An inhibitor may comprise aprotein, a peptide, a nucleic acid (such as an RNAi molecule orantisense RNA, including siRNA), or a small molecule.

As used herein, the term “depolarization” refers to a change in theelectrical potential difference across the cell membrane (between theinside of the cell and the outside of the cell, with outside taken asground potential), where that electrical potential difference isreduced, eliminated, or reversed in polarity. Activation of anon-selective channel, such as the NC_(Ca-ATP) channel, will typicallyincrease in the permeability of the cell membrane to sodium and otherions effective to reduce the magnitude, and may nearly or completelyeliminate, the electrical potential difference across a cell membrane.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are interchangeable and refer to an amount thatresults in an improvement or remediation of at least one symptom of thedisease or condition. Those of skill in the art understand that theeffective amount may improve the patient's or subject's condition, butmay not be a complete cure of the disease and/or condition.

As used herein, the term “endothelium” refers to a layer of cells thatline the inside surfaces of body cavities, blood vessels, and lymphvessels or that form capillaries.

As used herein, the term “endothelial cell” refers to a cell of theendothelium or a cell that lines the surfaces of body cavities, forexample, blood or lymph vessels or capillaries. In certain embodiments,the term endothelial cell refers to a neural endothelial cell or anendothelial cell that is part of the nervous system, for example thecentral nervous system or the brain or spinal cord.

As used herein, the term “inhibit” refers to the ability of the compoundto block, partially block, interfere, decrease, reduce or deactivate achannel such as the NC_(Ca-ATP) channel. Thus, one of skill in the artunderstands that the term inhibit encompasses a complete and/or partialloss of activity of a channel, such as the NC_(Ca-ATP) channel. Channelactivity may be inhibited by channel block (occlusion or closure of thepore region, preventing ionic current flow through the channel), bychanges in an opening rate or in the mean open time, changes in aclosing rate or in the mean closed time, or by other means. For example,a complete and/or partial loss of activity of the NC_(Ca-ATP) channel asmay be indicated by a reduction in cell depolarization, reduction insodium ion influx or any other monovalent ion influx, reduction in aninflux of water, reduction in extravasation of blood, reduction in celldeath, as well as an improvement in cellular survival following anischemic challenge.

As used herein, the term “inhibits the NC_(Ca-ATP) channel” refers to areduction in, cessation of, or blocking of, the activity of theNC_(Ca-ATP) channel, including inhibition of current flow through thechannel, inhibition of opening of the channel, inhibition of activationof the channel, inhibition or reduction of the expression of thechannel, including inhibition or reduction of genetic message encodingthe channel and inhibition or reduction of the production channelproteins, inhibition or reduction of insertion of the channel into theplasma membrane of a cell, or other forms of reducing the physiologicactivity of the NC_(Ca-ATP) channel.

The term “morbidity” as used herein is the state of being diseased. Yetfurther, morbidity can also refer to the disease rate or the ratio ofsick subjects or cases of disease in to a given population.

The term “mortality” as used herein is the state of being mortal orcausing death. Yet further, mortality can also refer to the death rateor the ratio of number of deaths to a given population.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

As used herein, the term “reduces” refers to a decrease in cell death,inflammatory response, hemorrhagic conversion, extravasation of blood,etc. as compared to no treatment with the compound of the presentinvention. Thus, one of skill in the art is able to determine the scopeof the reduction of any of the symptoms and/or conditions associatedwith a spinal cord injury in which the subject has received thetreatment of the present invention compared to no treatment and/or whatwould otherwise have occurred without intervention.

As used herein, the terms “SUR1 antagonist,” “SUR1 inhibitor,” and “SUR1blocker” and their grammatical variants may be used interchangeably andeach refers to compounds that reduce the activity or effect of thereceptors SUR1, and include (but are not limited to) such compounds as,for example, glibenclamide (also known as glyburide), tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds(estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g.,diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone, etc.)and combinations thereof. Chemical names of some SUR1 antagonistsinclude: glibenclamide (1[p-2[5-chloro-O-anisamido)ethyl] phenyl]sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide (1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido) ethyl] phenyl] sulfonyl] urea); andtolazamide (benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl)

As used herein, the terms “TRPM4 antagonist,” “TRPM4 inhibitor,” and“TRPM4 blocker” and their grammatical variants may be usedinterchangeably and each refers to compounds that reduce the activity oreffect of the TRPM4 channel, e.g. by reducing or blocking the flow ofions through the TRPM4 pore, and include (but are not limited to) suchcompounds as, for example, pinkolant, rimonabant, a fenamate (such asflufenamic acid, mefenamic acid, meclofenamic acid, or niflumic acid),1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazolehydrochloride, and a biologically active derivatives thereof.

The terms “treating” and “treatment” as used herein refer toadministering to a subject a therapeutically effective amount of acomposition so that the subject has an improvement in the disease orcondition. The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient's condition, but may not be a complete cure of the disease.Treating may also comprise treating subjects at risk of developing adisease and/or condition.

II. Exemplary Embodiments of the Invention

In particular cases of the invention, there are methods and/orcompositions for the treatment and/or prevention of spinal cord injury,brain injury, and other damage to the nervous system, such as, e.g.,injury related to progressive hemorrhagic necrosis, and intraventricularhemorrhage.

A. Intraventricular Hemorrhage

In exemplary embodiments of the invention, there are methods andcompositions and kits for the treatment and/or prevention ofintraventricular hemorrhage in an individual. The present inventionconcerns a specific channel, the NC_(Ca-ATP) channel, which isexpressed, for example, in the vasculature endothelium and germinalmatrix following intraventricular hemorrhage (IVH). This uniquenon-selective cation channel is activated by intracellular calcium andblocked by intracellular ATP (NC_(Ca-ATP) channel), and can be also beexpressed in, for example, neural cells, such as neuronal cells,neuroglia cells (also termed glia, or glial cells, e.g., astrocyte,ependymal cell, oligodentrocyte and microglia) or endothelial cells(e.g., capillary endothelial cells) in which the cells have been or areexposed to a traumatic insult, for example, an acute insult (e.g.,hypoxia, ischemia, tissue compression, mechanical distortion, cerebraledema or cell swelling), toxic compounds or metabolites, an acuteinjury, cancer, and brain abscess.

Without being bound by theory, it is believed that the hypoxic-ischemicenvironment in prematurity leads to transcriptional activation of SUR1and opening of NC(Ca-ATP) channels in IVH, initiating a cascade ofevents culminating in acute hemorrhage in parallel with ischemic stroke.

Intraventricular hemorrhage is bleeding into ventricular spaces, whichare spaces in the brain that carry cerebrospinal fluid. Following birth,the premature infant's brain is exposed to changes in blood flow andoxygen levels, which may cause the many tiny, fragile blood vessels ofthe infant's brain to break and bleeding to occur. Such an event happensusually in babies who are extremely premature or who have medicalproblems during or after birth. Intraventricular hemorrhage often occursin very low birthweight babies weighing less than 1,500 grams. Almostall IVH occurs within the first week of life.

Babies with respiratory problems such as hyaline membrane disease, orother complications of prematurity, are at greater risk to have IVH. Thesmaller and more premature the baby, the more likely IVH will occur.Although many babies have no symptoms at the time that bleeding occurs,some infants do have symptoms, including apnea, bradycardia, poor muscletone, decreased activity, anemia, seizures, high-pitched cry, weak suck,cyanosis, and/or bulging fontanel.

Infants at risk for IVH may have an ultrasound of the head to look forbleeding in the first days following birth. IVH is graded on a scale ofone to four, with grade IV being most severe. Grade 1 is considered whenbleeding occurs just in a small area of the ventricles; in Grade 2,bleeding also occurs inside the ventricles; in Grade 3, ventricles areenlarged by the blood; and in Grade 4, there is bleeding into the braintissues around the ventricles.

More than half of babies born weighing less than 1,000 grams haveintraventricular hemorrhages, although most of these bleeds are mild(Grade I or II), and many resolve with few or no problems, wherein, forexample, the body absorbs the blood. In more severe cases (Grade III orIV), however, as blood absorbs there can be damage to the brain tissue,and these cases (especially Grade IV) can result in additional problems,such as enlarged ventricles, hydrocephalus, cerebral palsy, hearingloss, vision problems, and/or learning disabilities, for example.

In some cases, the infant develops hydrocephalus, which may be treatedby medicines to decrease the amount of spinal fluid that the brainmakes, frequent lumbar punctures (LPs), reservoir, or shunt.

Long-term abnormalities that may occur following intraventricularhemorrhage include at least motor (movement) problems (tight or stiffmuscles; slow to crawl, stand, or walk; abnormal crawling, toe walking;moving one side more than the other; frequent arching of the back (notjust when angry or at play); slow mental development (does not listen tothe parent voice by age 3-4 months after hospital discharge; does notmake different sounds by 8-9 months after discharge; does not seem tounderstand or say any words by 12-13 months after discharge); seizure;deafness; blindness; poor coordination or balance; specific learningdisabilities (math or reading); very short attention span; behavioralproblems; difficulty with activities that require coordination of theeyes and hands, for example, catching a ball or copying a simpledrawing; and vision correction, for example.

Prior to the present invention, there was no treatment forintraventricular hemorrhage itself, although mother's between 24 and 34weeks of gestation and may be at risk for early delivery may be providedcorticosteroids before delivery, which has been shown to lower the riskof IVH in the baby.

In other embodiments, the present invention is drawn to the regulationand/or modulation of this NC_(Ca-ATP) channel and how its modulation canbe used to treat various diseases and/or conditions, for example, IVH.In specific embodiments, the modulation and/or regulation of the channelresults from administration of an antagonist or inhibitor of thechannel. Thus, depending upon the disease state or progression, acomposition (an antagonist or inhibitor) is administered to block orinhibit at least in part the channel to prevent cell death, for example,that results from IVH. In these instances, the channel is blocked toprevent or reduce or modulate, for example, depolarization of the cellsor other pathological conditions associated with IVH.

In one aspect, the present invention provides novel methods of treatinga patient comprising administering at least a therapeutic compound thattargets a unique non-selective cation channel activated by intracellularcalcium and blocked by intracellular ATP (NC_(Ca-ATP) channel), incombination with an additional therapeutic compound. In specificembodiments, the therapeutic compound that targets the channel may be anantagonist (such as a SUR1 inhibitor or a TRPM4 inhibitor, for example)that is employed in therapies, such as treatment of IVH, wherebyblocking and/or inhibiting the NC_(Ca-ATP) channel amelioratespathological conditions associated with IVH.

In certain embodiments, additional compounds for the compositions of theinvention include cation channel blockers and antagonists of VEGF, MMP,NOS, and/or thrombin, for example.

Further embodiments comprises a method of treating a subject at risk ofIVH comprising administering to the subject a combinatorial therapeuticcomposition effective at least in part to inhibit a NC_(Ca-ATP) channelin a cell, such as, for example, an endothelial cell, germinal matrixtissue, or a combination thereof.

The invention also encompasses the use of such compounds incombinatorial compositions that at least in part modulate NC_(Ca-ATP)channel activity to treat, for example, IVH. In certain embodiments, IVHcauses cell swelling resulting in cellular damage (including, forexample, cell death). Further provided by the invention is a method ofpreventing cellular swelling and the resulting cellular damage throughthe therapeutic use of antagonists to the NC_(Ca-ATP) channel, incombination with an additional therapeutic compound. In one embodiment,the therapeutic combinatorial composition can be administered to apremature infant subject to or undergoing IVH. The invention furtherprovides the therapeutic use of sulfonylurea compounds as antagonists tothe NC_(Ca-ATP) channel to treat IVH. In one embodiment the sulfonylureacompound is glibenclamide. In another embodiment, the sulfonylureacompound is tolbutamide, or any of the other compounds that have beenfound to promote insulin secretion by acting on KATP channels inpancreatic β cells, as listed elsewhere herein.

In certain embodiments, NC_(Ca-ATP) channel is blocked, inhibited, orotherwise is decreased in activity. In such examples, an antagonist ofthe NC_(Ca-ATP) channel is administered and/or applied. The antagonistmodulates the NC_(Ca-ATP) channel such that flux (ion and/or water)through the channel is reduced, ceased, decreased and/or stopped. Theantagonist may have a reversible or an irreversible activity withrespect to the activity of the NC_(Ca-ATP) channel IVH. Thus, inhibitionof the NC_(Ca-ATP) channel can reduce cytotoxic edema and death ofendothelial cells which are associated IVH.

Accordingly, the present invention is useful in the treatment orprevention of IVH. According to a specific embodiment of the presentinvention the administration of effective amounts of the active compoundcan block the channel, which if remained open leads to cell swelling andcell death. A variety of antagonists to SUR1 are suitable for blockingthe channel. Examples of suitable SUR1 antagonists include, but are notlimited to glibenclamide, tolbutamide, repaglinide, nateglinide,meglitinide, midaglizole, LY397364, LY389382, glyclazide, glimepiride,estrogen, estrogen related-compounds and combinations thereof. In apreferred embodiment of the invention the SUR1 antagonists is selectedfrom the group consisting of glibenclamide and tolbutamide. Anotherantagonist that can be used is MgADP. Still other therapeutic“strategies” for preventing cell swelling and cell death can be adoptedincluding, but not limited to methods that maintain the cell in apolarized state and methods that prevent strong depolarization.

In certain embodiments, the invention encompasses antagonists of theNC_(Ca-ATP) channel, including small molecules, large molecules, andantibodies, as well as nucleotide sequences that can be used to inhibitNC_(Ca-ATP) channel gene expression (e.g., antisense and ribozymemolecules). An antagonist of the NC_(Ca-ATP) channel includes one ormore compounds capable of (1) blocking the channel; (2) preventingchannel opening; (3) reducing the magnitude of membrane current throughthe channel; (4) inhibiting transcriptional expression of the channel;and/or (5) inhibiting post-translational assembly and/or trafficking ofchannel subunits.

In certain embodiments of the invention, several pathways to cell deathare involved in IVH, which require monovalent or divalent cation influx,implicating non-selective cation (NC) channels. In specific embodiments,NC channels are also likely to be involved in the dysfunction ofvascular endothelial cells that leads to formation of edema IVH. Inother specific embodiments, blockers of NC channels, includingpinokalant (LOE 908 MS) and rimonabant (SR141716A) can be administeredto treat IVH.

In other embodiments of the invention, IVH causes capillary dysfunction,resulting in edema formation and hemorrhagic conversion. In specificembodiments, the invention generally concerns the central role ofStarling's principle, which states that edema formation is determined bythe “driving force” and capillary “permeability pore.” In particularaspects related to the invention, movements of fluids are driven largelywithout new expenditure of energy. In one embodiment, the progressivechanges in osmotic and hydrostatic conductivity of abnormal capillariesis organized into 3 phases: formation of ionic edema, formation ofvasogenic edema, and catastrophic failure with hemorrhagic conversion.In certain embodiments, IVH capillary dysfunction is attributed to denovo synthesis of a specific ensemble of proteins that determine theterms for osmotic and hydraulic conductivity in Starling's equation, andwhose expression is driven by a distinct transcriptional program.

Another embodiment of the present invention comprises a method ofreducing morbidity and morality of a subject suffering from IVHcomprising administering to the subject a therapeutic compositioncomprising a single NC_(Ca-ATP) channel inhibitor or a combinatorialtherapeutic composition effective to inhibit NC_(Ca-ATP) channels in acell, including, for example, an endothelial cell, germinal matrixtissue, or a combination thereof. In specific embodiments, morbidity andmortality includes, for example, death, shunt-dependent hydrocephalus,and life-long neurological consequences such as cerebral palsy,seizures, mental retardation, and other neurodevelopmental disabilities.

In specific embodiments, the individual is an infant, including apremature infant, although in alternative embodiments the individual isa child or adult. The treatment and/or prevention may occur prior and/orfollowing birth of the infant, and the treatment and/or prevention maybe directed to the mother during pregnancy, in specific embodiments. Inparticular cases, the pregnant mother is at risk for deliveryprematurely and may be provided methods and compositions of theinvention to treat and/or prevent intraventricular hemorrhage in theinfant following birth. Women at risk for preterm delivery include atleast if they have one or more of the following conditions orsituations: pregnant with multiples; have had a previous prematurebirth; have certain uterine or cervical abnormalities; recurring bladderand/or kidney infections; urinary tract infections, vaginal infections,and sexually transmitted infections; infection with fever (greater than101 degrees F.) during pregnancy; unexplained vaginal bleeding after 20weeks of pregnancy; chronic illness such as high blood pressure, kidneydisease or diabetes; multiple first trimester abortions or one or moresecond trimester abortions; underweight or overweight before pregnancy;clotting Disorder (thrombophilia); being Pregnant with a single fetusafter in vitro fertilization (IVF); short time between pregnancies (lessthan 6-9 months between birth and beginning of the next pregnancy);little or no prenatal care; smoking; drinking alcohol; using illegaldrugs; victim of domestic violence, including physical, sexual oremotional abuse; lack of social support; high levels of stress; lowincome; and/or long working hours with long periods of standing.

Thus, in women at risk for preterm delivery, the mother or infant (inutero) may be provided methods and/or compositions of the invention,including women at risk for developing premature labor or who havesymptoms of having premature labor, such as having labor symptoms priorto 37 weeks of gestation. Alternatively, or in addition, the inventivemethods and/or compositions may be provided to the infant followingbirth.

The treatment and/or prevention of intraventricular hemorrhage utilizesinhibitors of a NC_(Ca-ATP) channel, and in particular cases thischannel is upregulated in brain tissues prior to and/or during onset ofintraventricular hemorrhage. In certain aspects, the channel isupregulated in endothelial cells in the brain, neural cells, includingneuronal cells, and so forth. In specific embodiments, the inhibitorsare directed to a regulatory component of the channel and/or apore-forming subunit of the channel, although other components of thechannel may be targeted, or example. The inhibitors, in particularcases, are directed to SUR1, a regulatory subunit of the channel. TRPM4,a pore-forming subunit of the channel, or they may be mixtures orcombinations thereof. SUR1 inhibitors include sulfonylurea compounds,benzamido derivatives, or mixtures thereof.

In a specific embodiment, the inhibitor is provided to the mother priorto 37 weeks of gestation. In another specific embodiment, the mother isat risk for premature labor. In a further specific embodiment, thepregnancy is less than 37 weeks in gestation and the mother has one ormore symptoms of labor. Symptoms of labor are known in the art, althoughin specific embodiments they include one or more of the following: acontraction every 10 minutes, or more frequently within one hour (fiveor more uterine contractions in an hour); watery fluid leaking from thevagina, which could signal that the bag of water has broken;menstrual-like cramps felt in the lower abdomen that may be transient orconstant; low, dull backache experienced below the waistline that may betransient or constant; pelvic pressure; abdominal cramps that may occurwith or without diarrhea; and/or increase or change in vaginaldischarge.

B. Spinal Cord Injury and Progressive Hemorrhagic Necrosis

Acute spinal cord injury (SCI) results in progressive hemorrhagicnecrosis (PHN), a poorly understood pathological process characterizedby hemorrhage and necrosis that leads to devastating loss of spinal cordtissue, cyctic cavitation of the cord, and debilitating neurologicaldysfunction. Using a rodent model of severe cervical SCI, SUR1-regulatedNC_(Ca-ATP) channels were characterized for involvement in PHN. Incontrols, SCI caused a progressively expansive lesion with fragmentationof capillaries, hemorrhage that doubled in volume over 12 h, tissuenecrosis and severe neurological dysfunction. Necrotic lesions weresurrounded by widespread up-regulation of SUR1 in capillaries andneurons. Patch clamp of cultured endothelial cells exposed to hypoxiashowed that up-regulation of SUR1 was associated with expression offunctional SUR1-regulated NC_(Ca-ATP) channels. Following SCI, block ofSUR1 by glibenclamide or repaglinide, or gene suppression of SUR1 byphosphorothioated antisense oligodeoxynucleotide, essentially eliminatedcapillary fragmentation and progressive accumulation of blood, wasassociated with significant sparing of white matter tracts and a 3-foldreduction in lesion volume, and resulted in marked neurobehavioralfunctional improvement compared to controls. Therefore, SUR1-regulatedNC_(Ca-ATP) channels in capillary endothelium are critical todevelopment of PHN and constitute a major novel target for therapy inSCI.

1. Spinal Cord Injury—the Clinical Problem

Acute spinal cord injury (SCI) results in physical disruption of spinalcord neurons and axons leading to deficits in motor, sensory, andautonomic function. This is a debilitating neurological disorder commonin young adults that often requires life-long therapy and rehabilitativecare, placing a significant burden on healthcare systems. The fact thatSCI impacts mostly young people makes the tragedy all the more horrific,and the cost to society in terms of lost “person-years” all the moreenormous. Sadly, many patients exhibit neuropathologically andclinically complete cord injuries following SCI. However, many othershave neuropathologically incomplete lesions (Hayes and Kakulas, 1997;Tator and Fehlings, 1991). giving hope that proper treatment to minimizesecondary injury may reduce the functional impact.

2. Secondary Injury—Progressive Hemorrhagic Necrosis (PHN)

The concept of secondary injury in SCI arises from the observation thatthe volume of injured tissue increases with time after injury, i.e., thelesion itself expands and evolves over time. Whereas primary injuredtissues are irrevocably damaged from the very beginning, right afterimpact, tissues that are destined to become “secondarily” injured areconsidered to be potentially salvageable. Secondary injury in SCI hasbeen reviewed in a classic paper by Tator (1991), as well as in morerecent reviews (Kwon et al., 2004), wherein the overall concept ofsecondary injury is validated. Older observations based on histologicalstudies that gave rise to the concept of lesion-evolution have beenconfirmed with non-invasive MRI (Bilgen et al., 2000; Ohta et al., 1999;Sasaki et al., 1978; Weirich et al., 1990).

Numerous mechanisms of secondary injury are recognized, including edema,ischemia, oxidative stress and inflammation. In SCI, however, onepathological entity in particular is recognized that is relativelyunique to the spinal cord and that has especially devastatingconsequences—progressive hemorrhagic necrosis (PHN) (Fitch et al., 1999;Kraus, 1996; nelson et al., 1977; Tator, 1991; Tator and Fehlings, 1991;Tator and Koyanagi, 1997).

PHN is a rather mysterious condition, first recognized over 3 decadesago, that has previously eluded understanding and treatment. Asdisclosed herein, the present invention provides treatment for thiscondition. Following impact, petechial hemorrhages form in surroundingtissues and later emerge in more distant tissues, eventually coalescinginto the characteristic lesion of hemorrhagic necrosis. The specifictime course and magnitude of these changes remain to be determined, butpapers by Khan et al. (1985) and Kawata et al. (1993) nicely describethe progressive increase in hemorrhage in the cord. After injury, asmall hemorrhagic lesion involving primarily the capillary-rich centralgray matter is observed at 15 min, but hemorrhage, necrosis and edema inthe central gray matter enlarge progressively over a period of 3-24 h(Balentine, 1978; Iizuka et al., 1987; Kawata et al., 1993). The whitematter surrounding the hemorrhagic gray matter shows a variety ofabnormalities, including decreased H&E staining, disrupted myelin, andaxonal and periaxonal swelling. Tator and Koyanagi (1997) noted thatwhite matter lesions extend far from the injury site, especially in theposterior columns. The evolution of hemorrhage and necrosis has beenreferred to as “autodestruction”, and it is this that forms the keyobservation that defines PHN. PHN eventually causes loss of vital spinalcord tissue and, in some species including humans, leads topost-traumatic cystic cavitation surrounded by glial scar tissue.

3. Mechanisms of Delayed Hemorrhage and PHN

Tator and Koyanagi (1997) expressed the view that obstruction of smallintramedullary vessels by the initial mechanical stress or secondaryinjury may be responsible for PHN. Kawata and colleagues (1993)attributed the progressive changes to leukocyte infiltration around theinjured area leading to plugging of capillaries. Most importantly,damage to the endothelium of spinal cord capillaries and postcapillaryvenules has been regarded as a major factor in the pathogenesis of PHN(Griffiths et al., 1978; Kapadia, 1984; Nelson et al., 1977). Thatendothelium is involved is essentially certain, given that petechialhemorrhages, the primary characteristic of PHN, arise from nothing lessthan catastrophic failure of capillary or venular integrity. However, nomolecular mechanism for progressive dysfunction of endothelium hasheretofore been identified.

“Hemorrhagic conversion” is a term familiar to many from the strokeliterature, but not from the SCI literature. Hemorrhagic conversiondescribes the process of conversion from a bland infarct into ahemorrhagic infarct, and is typically associated with post-ischemicreperfusion, either spontaneous or induced by thrombolytic therapy. Themolecular pathology involved in hemorrhagic conversion has yet to befully elucidated, but considerable work has implicated enzymaticdestruction of capillaries by matrix-metalloproteinases (MMP) releasedby invading neutrophils (Gidday et al., 2005; Justicia et al., 2003;Lorenzl et al., 2003; Romanic et al., 1998). Maladaptive activation ofMMP compromises the structural integrity of capillaries, leading toformation of petechial hemorrhages. In ischemic stroke, MMP inhibitorsreduce hemorrhagic conversion following thrombolytic-inducedreperfusion. MMPs are also implicated in spinal cord injury (de et al.,2000; Duchossoy et al., 2001; Duchossoy et al., 2001; Goussev et al.,2003; Hsu et al., 2006; Noble et al., 2002; Wells et al., 2003). In SCI,however, their role has been studied predominantly in the context ofdelayed tissue healing, and no evidence has been put forth to suggesttheir involvement in PHN.

Expression and activation of NC_(Ca-ATP) channels (see Simard et al.,2007) gives rise to PHN. The data demonstrate that cells that expressthe NC_(Ca-ATP) channel following an ischemic or other injury-stimulus,later undergo oncotic (necrotic) cell death when ATP is depleted. Thisis shown explicitly for astrocytes (Simard et al., 2006), and inspecific embodiments it also occurs with capillary endothelial cellsthat express the channel. It follows that if capillary endothelial cellsundergo this process leading to necrotic death, capillary integritywould be lost, leading to extravasation of blood and formation ofpetechial hemorrhages. Applicants disclose herein that inhibition ofNC_(Ca-ATP) channels is useful to prevent and to treat PHN and SCI.

4. Therapies in SCI

No cure exists for the primary injury in SCI, but research hasidentified various pharmacological compounds that specificallyantagonize secondary injury mechanisms responsible for worsened outcomein SCI. Several compounds including methylprednisolone, GM-1ganglioside, thyrotropin releasing hormone, nimodipine, and gacyclidinehave been tested in prospective randomized clinical trials of SCI, withonly methylprednisolone and GM-1 ganglioside showing evidence of amodest benefit (Fehlings and Baptiste, 2005). At present, high dosemethylprednisolone steroid therapy is the only pharmacological therapyshown to have efficacy in a Phase Three randomized trial when it can beadministered within eight hours of injury (Bracken, 2002; Bracken etal., 1997; Bracken et al., 1998).

Of the numerous treatments assessed in SCI, very few have been shown toactually decrease the hemorrhage and tissue loss associated with PHN.Methylprednisolone, the only approved therapy for SCI, improves edema,but does not alter the development of PHN (Merola et al., 2002). Anumber of compounds have shown beneficial effects related to sparing ofwhite matter, including the NMDA antagonist, MK801 (Faden et al., 1988),the AMPA antagonist, GYKI 52466 (Colak et al., 2003), Na⁺ channelblockers (Schwartz and Fehlings, 2001; Teng and Wrathall, 1997),minocycline (Teng et al., 2004), and estrogen (Chaovipoch et al., 2006).

However, no treatment has been previously reported that reduces PHN andlesion volume, and that improves neurobehavioral function to the extentthat is disclosed herein in which the highly selective but exemplarySUR1 antagonists, glibenclamide and repaglinide, as well as withantisense-oligodeoxynucleotide (AS-ODN) directed against SUR1, are ableto treat PHN. It is useful that the molecular mechanisms targeted bythese 3 agents—SUR1 and the SUR1-regulated NC_(Ca-ATP) channel, arecharacterized to further elucidate their role in PHN.

III. NC_(Ca-ATP) Channel

A unique non-selective monovalent cationic ATP-sensitive channel(NC_(Ca-ATP) channel) was identified first in native reactive astrocytes(NRAs) and later in neurons and capillary endothelial cells after strokeor traumatic brain or spinal cord injury (see International applicationWO 03/079987 to Simard et al., and Chen and Simard, 2001, eachincorporated by reference herein in its entirety). As with the K_(ATP)channel in pancreatic β cells, the NC_(CaATP) channel is considered tobe a heteromultimer structure comprised of sulfonylurea receptor type 1(SUR1) regulatory subunits and pore-forming subunits (Chen et al.,2003), which include TRPM4 pore subunits.

The invention is based, in part, on the discovery of a specific channel,the NC_(Ca-ATP) channel, defined as a channel on astrocytes in U.S.Application Publication No. 20030215889, which is incorporated herein byreference in its entirety. More specifically, the present invention hasfurther defined that this channel is not only expressed on astrocytes,it is expressed at least on neural cells, neuroglial cells, and/orneural endothelial cells after brain and spinal cord trauma, forexample, an hypoxic event, an ischemic event, or other secondaryneuronal injuries relating to these events.

The NC_(Ca-ATP) channel is activated by calcium ions (Ca²⁺) and issensitive to ATP. Thus, this channel is a non-selective cation channelactivated by intracellular Ca²⁺ and blocked by intracellular ATP. Whenopened by depletion of intracellular ATP, this channel is responsiblefor complete depolarization due to massive Na⁺ influx, which creates anelectrical gradient for Cl⁻ and an osmotic gradient for H₂O, resultingin cytotoxic edema and cell death. When the channel is blocked orinhibited, massive Na⁺ does not occur, thereby preventing cytotoxicedema.

Certain functional characteristics distinguish the NC_(Ca-ATP) channelfrom other known ion channels. These characteristics can include, butare not limited to, at least some of the following: 1) it is anon-selective cation channel that readily allows passage of Na⁺, K⁺ andother monovalent cations; 2) it is activated by an increase inintracellular calcium, and/or by a decrease in intracellular ATP; 3) itis regulated by sulfonylurea receptor type 1 (SUR1), which heretoforehad been considered to be associated exclusively with K_(ATP) channelssuch as those found in pancreatic β cells.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, whereconcentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channel isalso inhibited by cytoplasmic ATP in a physiological concentrationrange, where the concentration range is about 0.1 mM to about 10 mM, ormore particularly about 0.2 mM to about 5 mM. The NC_(Ca-ATP) channel isalso permeable to the following cations; K⁺, Cs⁺, Li⁺, Na⁺; to theextent that the permeability ratio between any two of the cations isgreater than 0.5 and less than 2.

SUR imparts sensitivity to antidiabetic sulfonylureas such asglibenclamide and tolbutamide and is responsible for activation by achemically diverse group of agents termed “K⁺ channel openers” such asdiazoxide, pinacidil and cromakalin (Aguilar-Bryan et al., 1995; Inagakiet al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Shyng et al.,1997). In various tissues, molecularly distinct SURs are coupled todistinct pore-forming subunits to form different K_(ATP) channels withdistinguishable physiological and pharmacological characteristics. TheK_(ATP) channel in pancreatic f cells is formed from SUR1 linked withKir6.2, whereas the cardiac and smooth muscle K_(ATP) channels areformed from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively(Fujita et al., 2000). Despite being made up of distinctly differentpore-forming subunits, the NC_(Ca-ATP) channel is also sensitive tosulfonylurea compounds.

Also, unlike the K_(ATP) channel, the NC_(Ca-ATP), channel conductssodium ions, potassium ions, cesium ions and other monovalent cationswith near equal facility (Chen and Simard, 2001) suggesting further thatthe characterization, and consequently the affinity to certaincompounds, of the NC_(Ca-ATP) channel differs from the K_(ATP) channel.

Other nonselective cation channels that are activated by intracellularCa²⁺ and inhibited by intracellular ATP have been identified by othersbut not in astrocytes or neurons as disclosed herein. Further, theNC_(Ca-ATP), channel expressed and found in astrocytes differsphysiologically from the other channels with respect to calciumsensitivity and adenine nucleotide sensitivity (Chen et al., 2001).

The NC_(Ca-ATP) channel can be inhibited by an NC_(Ca-ATP) channelinhibitor, an NC_(Ca-ATP) channel blocker, a type 1 sulfonylureareceptor (SUR1) antagonist, SUR1 inhibitor, or a compound capable ofreducing the magnitude of membrane current through the channel. Morespecifically, the exemplary SUR1 antagonist may be selected from thegroup consisting of glibenclamide, tolbutamide, repaglinide,nateglinide, meglitinide, midaglizole, LY397364, LY389382, glyclazide,glimepiride, estrogen, estrogen related-compounds (estradiol, estrone,estriol, genistein, non-steroidal estrogen (e.g., diethystilbestrol),phytoestrogen (e.g., coumestrol), and zearalenone), and compounds knownto inhibit or block K_(ATP) channels. MgADP can also be used to inhibitthe channel. Other compounds that can be used to block or inhibitK_(ATP) channels include, but are not limited to tolbutamide, glyburide(1[[(p-2[5-chloro-O-anisamido)ethyl] phenyl]sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide (1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido)ethyl] phenyl] sulfonyl] urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]carbonyl]-4-methyl). In additional embodiments, non-sulfonyl ureacompounds, such as 2, 3-butanedione and 5-hydroxydecanoic acid, quinine,and therapeutically equivalent salts and derivatives thereof, may beemployed in the invention.

The channel is expressed on cells, including, for example, vascularendothelial cells and germinal matrix tissue. In specific embodiments,the inhibitor of the channel blocks the influx of Na+ into the cellsthereby preventing depolarization or other deleterious effects caused bythe altered ionic concentration of the cells. Inhibition of the influxof Na+ into the cells, thereby at least prevents or reduces cytotoxicedema and/or ionic edema. Thus, this treatment reduces cell death,including, for example, necrotic cell death. In specific embodiments,the invention reduces cell death of endothelial cells.

The compound can be administered alimentarily (e.g., orally, buccally,rectally or sublingually); parenterally (e.g., intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneously, intraperitoneally, intraventricularly); by intracavity;intravesically; intrapleurally; and/or topically (e.g., transdermally),mucosally, or by direct injection into the brain parenchyma.

Another embodiment of the present invention comprises a method oftreating a subject at risk for developing edema comprising administeringto the subject a therapeutic composition effective to inhibit aNC_(Ca-ATP) channel in at least an endothelial cell, germinal matrixtissue, or combination thereof. In specific embodiments, the compositionis effective to inhibit a NC_(Ca-ATP) channel in an endothelial cell.

In further embodiments, the compound that inhibits the NC_(Ca-ATP)channel can be administered in combination with one or more statins,diuretics, vasodilators (e.g., nitroglycerin), mannitol, diazoxide orsimilar compounds that stimulate or promote ischemic preconditioning.

Yet further, another embodiment of the present invention comprises apharmaceutical composition comprising or more statins, diuretics,vasodilators, mannitol, diazoxide or similar compounds that stimulate orpromote ischemic preconditioning or a pharmaceutically acceptable saltthereof and a compound that inhibits a NC_(Ca-ATP) channel or apharmaceutically acceptable salt thereof. This pharmaceuticalcomposition can be considered neuroprotective, in specific embodiments.For example, the pharmaceutical composition comprising a combination ofthe second agent and a compound that inhibits a NC_(Ca-ATP) channel istherapeutic or protective because it increases the therapeutic windowfor the administration of the second agent by several hours; for examplethe therapeutic window for administration of second agents may beincreased by several hours (e.g. about 4 to about 8 hrs) byco-administering antagonist of the NC_(Ca-ATP) channel.

An effective amount of a therapeutic composition of the invention,including an antagonist of NC_(Ca-ATP) channel and/or the additionaltherapeutic compound, that may be administered to a cell includes a doseof about 0.0001 nM to about 2000 μM, for example. More specifically,doses to be administered are from about 0.01 nM to about 2000 μM; about0.01 μM to about 0.05 μM; about 0.05 μM to about 1.0 μM; about 1.0 μM toabout 1.5 μM; about 1.5 μM to about 2.0 μM; about 2.0 μM to about 3.0μM; about 3.0 μM to about 4.0 μM; about 4.0 μM to about 5.0 μM; about5.0 μM to about 10 μM; about 10 μM to about 50 μM about 50 μM to about100 μM; about 100 μM to about 200 μM; about 200 μM to about 300 about300 to about 500 μM; about 500 to about 1000 μM; about 1000 μM to about1500 μM and about 1500 μM to about 2000 μM, for example. Of course, allof these amounts are exemplary, and any amount in-between these pointsis also expected to be of use in the invention.

An effective amount of an antagonist of the NC_(Ca-ATP) channel orrelated-compounds thereof as a treatment varies depending upon the hosttreated and the particular mode of administration. In one embodiment ofthe invention, the dose range of the therapeutic combinatorialcomposition of the invention, including an antagonist of NC_(Ca-ATP)channel and/or the additional therapeutic compound, will be about 0.01μg/kg body weight to about 20,000 ÿg/kg body weight. The term “bodyweight” is applicable when an animal is being treated. When isolatedcells are being treated, “body weight” as used herein should read tomean “total cell body weight”. The term “total body weight” may be usedto apply to both isolated cell and animal treatment. All concentrationsand treatment levels are expressed as “body weight” or simply “kg” inthis application are also considered to cover the analogous “total cellbody weight” and “total body weight” concentrations. However, those ofskill will recognize the utility of a variety of dosage range, forexample, 0.01 μg/kg body weight to 20,000 μg/kg body weight, 0.02 μg/kgbody weight to 15,000 μg/kg body weight, 0.03 μg/kg body weight to10,000 μg/kg body weight, 0.04 μg/kg body weight to 5,000 μg/kg bodyweight, 0.05 μg/kg body weight to 2,500 μg/kg body weight, 0.06 μg/kgbody weight to 1,000 μg/kg body weight, 0.07 μg/kg body weight to 500μg/kg body weight, 0.08 μg/kg body weight to 400 μg/kg body weight, 0.09μg/kg body weight to 200 μg/kg body weight or 0.1 μg/kg body weight to100 μg/kg body weight. Further, those of skill will recognize that avariety of different dosage levels will be of use, for example, 0.0001μg/kg, 0.0002 μg/kg, 0.0003 μg/kg, 0.0004 μg/kg, 0.005 μg/kg, 0.0007μg/kg, 0.001 μg/kg, 0.1 μg/kg, 1.0 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 5.0μg/kg, 10.0 μg/kg, 15.0 μg/kg, 30.0 μg/kg, 50 μg/kg, 75 μg/kg, 80 μg/kg,90 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg, 150 μg/kg, 160 μg/kg, 180μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg,350 μg/kg, 375 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600μg/kg, 700 μg/kg, 750 μg/kg, 800 ÿg/kg, 900 ÿg/kg, 1 mg/kg, 5 mg/kg, 10mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg.

In certain embodiments, there may be dosing of from very low ranges(e.g. 1 mg/kg/day or less; 5 mg/kg bolus; or 1 mg/kg/day) to moderatedoses (e.g. 2 mg bolus, 15 mg/day) to high doses (e.g. 5 mg bolus, 30-40mg/day; and even higher). Of course, all of these dosages are exemplary,and any dosage in-between these points is also expected to be of use inthe invention. Any of the above dosage ranges or dosage levels may beemployed for an agonist or antagonist, or both, of NC_(Ca-ATP) channelor related-compounds thereof.

In certain embodiments, the amount of the combinatorial therapeuticcomposition administered to the subject is in the range of about 0.0001μg/kg/day to about 20 mg/kg/day, about 0.01 μg/kg/day to about 100μg/kg/day, or about 100 μg/kg/day to about 20 mg/kg/day. Still further,the combinatorial therapeutic composition may be administered to thesubject in the form of a treatment in which the treatment may comprisethe amount of the combinatorial therapeutic composition or the dose ofthe combinatorial therapeutic composition that is administered per day(1, 2, 3, 4, etc.), week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5,etc.), etc. Treatments may be administered such that the amount ofcombinatorial therapeutic composition administered to the subject is inthe range of about 0.0001 μg/kg/treatment to about 20 mg/kg/treatment,about 0.01 μg/kg/treatment to about 100 μg/kg/treatment, or about 100μg/kg/treatment to about 20 mg/kg/treatment.

In another embodiment of the invention, there is a kit, housed in asuitable container, that comprises an inhibitor of NC_(Ca-ATP) channel.In another embodiment of the invention, the kit comprises an inhibitorof NC_(Ca-ATP) channel and, for example, one or more of a cation channelblocker and/or an antagonist of VEGF, MMP, NOS, or thrombin. The kit mayalso comprise suitable tools to administer compositions of the inventionto an individual.

The NC_(Ca-ATP) channel of the present invention is distinguished bycertain functional characteristics, the combination of whichdistinguishes it from known ion channels. The characteristics thatdistinguish the NC_(Ca-ATP) channel of the present invention include,but are not necessarily limited to, the following: 1) it is anon-selective cation channel that readily allows passage of Na, K andother monovalent cations; 2) it is activated by an increase inintracellular calcium, and/or by a decrease in intracellular ATP; 3) itis regulated by sulfonylurea receptor type 1 (SUR1), which heretoforehad been considered to be associated exclusively with K_(ATP) channelssuch as those found in pancreatic ÿ cells, for example.

More specifically, the NC_(Ca-ATP) channel of the present invention hasa single-channel conductance to potassium ion (K⁺) between 20 and 50 pS.The NC_(Ca-ATP) channel is also stimulated by Ca²⁺ on the cytoplasmicside of the cell membrane in a physiological concentration range, wheresaid concentration range is from 10⁻⁸ to 10⁻⁵ M. The NC_(Ca-ATP) channelis also inhibited by cytoplasmic ATP in a physiological concentrationrange, where said concentration range is from about 10⁻¹ mM to about 5mM. The NC_(Ca-ATP) channel is also permeable to the following cations;K⁺, Cs⁺, Li⁺, Na⁺; to the extent that the permeability ratio between anytwo of said cations is greater than 0.5 and less than 2.

IV. Exemplary Therapeutic and Preventative Embodiments

Treatment methods may involve treating an individual with an effectiveamount of a composition comprising an antagonist of NC_(Ca-ATP) channelor related-compound thereof. An effective amount is described,generally, as that amount sufficient to detectably and repeatedlyameliorate, reduce, minimize, limit the extent of a medical condition orits symptoms or, to prevent a disease or its medical condition. Morespecifically, it is envisioned that the treatment and/or prevention withan antagonist of NC_(Ca-ATP) channel or related-compounds thereof willinhibit cell depolarization, inhibit Na⁺ influx, inhibit an osmoticgradient change, inhibit water influx into the cell, inhibit cytotoxiccell edema, decrease stroke size, inhibit hemorrhagic conversion, and/ordecrease mortality of the subject, in specific embodiments

The effective amount of an antagonist of NC_(Ca-ATP) channel orrelated-compounds thereof to be used are those amounts effective toproduce beneficial results, for example, with respect to spinal cordinjury or progressive hemorrhagic necrosis treatment or prevention, inthe recipient animal or patient. Such amounts may be initiallydetermined by reviewing the published literature, by conducting in vitrotests and/or by conducting metabolic studies in healthy experimentalanimals, for example, as is routine in the art. Before use in a clinicalsetting, it may be beneficial to conduct confirmatory studies in ananimal model, preferably a widely accepted animal model of theparticular disease to be treated. Preferred animal models for use incertain embodiments are rodent models, which are preferred because theyare economical to use and, particularly, because the results gained arewidely accepted as predictive of clinical value.

As is well known in the art, a specific dose level of active compoundssuch as an antagonist of the NC_(Ca-ATP) channel or related-compoundsthereof for any particular patient depends upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the severity ofthe particular disease undergoing therapy. The person responsible foradministration will determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

One of skill in the art realizes that the effective amount of theantagonist or related-compound thereof can be the amount that isrequired to achieve the desired result: reduction in the risk of spinalcord injury or progressive hemorrhagic necrosis, reduction in the amountof damage following spinal cord injury or progressive hemorrhagicnecrosis, reduction in cell death, and so forth In specific embodiments,this amount also is an amount that maintains a reasonable level of bloodglucose in the patient, for example, the amount of the antagonistmaintains a blood glucose level of at least 60 mmol/l, more preferably,the blood glucose level is maintained in the range of about 60 mmol/l toabout 150 mmol/l. Thus, the amounts prevents the subject from becominghypoglycemic. If glucose levels are not normal, then one of skill in theart would administer either insulin or glucose, depending upon if thepatient is hypoglycemic or hyperglycemic.

Administration of the therapeutic antagonist of NC_(Ca-ATP) channelcomposition of the present invention to a patient or subject will followgeneral protocols for the administration of therapies used in spinalcord injury or progressive hemorrhagic necrosis treatment, taking intoaccount the toxicity, if any, of the antagonist of the NC_(Ca-ATP)channel. It is expected that the treatment cycles would be repeated asnecessary. It also is contemplated that various standard therapies, aswell as surgical intervention, may be applied in combination with thedescribed therapy.

Another aspect of the present invention for the treatment of IVH orspinal cord injury or progressive hemorrhagic conversion comprisesadministration of an effective amount of a SUR1 antagonist and/or aTRPM4 antagonist and administration of glucose. Glucose administrationmay precede the time of treatment with an antagonist of the NC_(Ca-ATP)channel, may be at the time of treatment with an antagonist of theNC_(Ca-ATP) channel, such as a SUR1 and/or TRPM4 antagonist, or mayfollow treatment with an antagonist of the NC_(Ca-ATP) channel (e.g., at15 minutes after treatment with an antagonist of the NC_(Ca-ATP)channel, or at one half hour after treatment with an antagonist of theNC_(Ca-ATP) channel, or at one hour after treatment with an antagonistof the NC_(Ca-ATP) channel, or at two hours after treatment with anantagonist of the NC_(Ca-ATP) channel, or at three hours after treatmentwith an antagonist of the N_(Ca-ATP) channel, for example). Glucoseadministration may be by intravenous, or intraperitoneal, or othersuitable route and means of delivery. Additional glucose allowsadministration of higher doses of an antagonist of the NC_(Ca-ATP)channel than might otherwise be possible, so that combined glucose withan antagonist of the NC_(Ca-ATP) channel provides greater protection,and may allow treatment at later times, than with an antagonist of theNC_(Ca-ATP) channel alone. Greater amounts of glucose are administeredwhere larger doses of an antagonist of the NC_(Ca-ATP) channel areadministered.

Yet further, the compositions of the present invention can be used toproduce neuroprotective kits that are used to treat subjects at risk orsuffering from conditions that are associated with spinal cord injury,including progressive hemorrhagic necrosis, for example.

V. Combinatorial Therapeutic Compositions

In certain embodiments of the present invention includes a combinatorialtherapeutic composition comprising an antagonist of the NC_(Ca-ATP)channel and another therapeutic compound, such as a cation channelblocker and/or an antagonist of a specific molecule, such as VEGF, MMP,NOS, thrombin, and so forth.

A. Inhibitors of NC_(Ca-ATP) Channel

According to a specific embodiment of the present invention, theadministration of effective amounts of the active compound can block thechannel, which if it remained open would lead cell swelling and celldeath. A variety of antagonists to SUR1 are suitable for blocking thechannel. Examples of suitable SUR1 antagonists include, but are notlimited to glibenclamide, tolbutamide, repaglinide, nateglinide,meglitinide, midaglizole, LY397364, LY3 89382, gliclazide, glimepiride,MgADP, and combinations thereof. In a preferred embodiment of theinvention the SUR1 antagonists is selected from the group consisting ofglibenclamide and tolbutamide. A variety of TRPM4 antagonists aresuitable for blocking the channel. Examples of suitable TRPM4antagonists include, but are not limited to, pinkolant, rimonabant, afenamate (such as flufenamic acid, mefenamic acid, meclofenamic acid, orniflumic acid),1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazolehydrochloride, and a biologically active derivative thereof. Still othertherapeutic “strategies” for preventing cell swelling and cell death canbe adopted including, but not limited to methods that maintain the cellin a polarized state and methods that prevent strong depolarization.

The present invention comprises modulators of the channel, for exampleone or more agonists and/or one or more antagonists of the channel.Examples of antagonists or agonists of the present invention mayencompass respective antagonists and/or agonists identified in USApplication Publication No. 20030215889, which is incorporated herein byreference in its entirety. One of skill in the art is aware that theNC_(Ca-ATP) channel is comprised of at least two subunits: theregulatory subunit, SUR1, and the pore forming subunit.

1. Exemplary SUR1 Inhibitors

In certain embodiments, antagonists to sulfonylurea receptor-1 (SUR1)are suitable for blocking the channel. Examples of suitable SUR1antagonists include, but are not limited to glibenclamide, tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds estrogenrelated-compounds (estradiol, estrone, estriol, genistein, non-steroidalestrogen (e.g., diethystilbestrol), phytoestrogen (e.g., coumestrol),zearalenone, etc.) and combinations thereof. In a preferred embodimentof the invention the SUR1 antagonists is selected from the groupconsisting of glibenclamide and tolbutamide. Yet further, anotherantagonist can be MgADP. Other antagonist include blockers of KATPchannels, for example, but not limited to tolbutamide, glibenclamide(1[p-2[5-chloro-O-anisamido)ethyl] phenyl]sulfonyl]-3-cyclohexyl-3-urea); chlopropamide(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide(1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido) ethyl] phenyl]sulfonyl] urea); ortolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1 yl)amino]carbonyl]-4-methyl).

2. Modulators of SUR1 Transcription and/or Translation

In certain embodiments, the modulator can comprise a compound (protein,nucleic acid, siRNA, etc.) that modulates transcription and/ortranslation of SUR1 (regulatory subunit) and/or the molecular entitiesthat comprise the pore-forming subunit.

3. Transcription Factors

Transcription factors are regulatory proteins that binds to a specificDNA sequence (e.g., promoters and enhancers) and regulate transcriptionof an encoding DNA region. Thus, transcription factors can be used tomodulate the expression of SUR1. Typically, a transcription factorcomprises a binding domain that binds to DNA (a DNA-binding domain) anda regulatory domain that controls transcription. Where a regulatorydomain activates transcription, that regulatory domain is designated anactivation domain. Where that regulatory domain inhibits transcription,that regulatory domain is designated a repression domain. Morespecifically, transcription factors such as Sp1, HIF1, and NFB can beused to modulate expression of SUR1.

In particular embodiments of the invention, a transcription factor maybe targeted by a composition of the invention. The transcription factormay be one that is associated with a pathway in which SUR1 is involved.The transcription factor may be targeted with an antagonist of theinvention, including siRNA to downregulate the transcription factor.Such antagonists can be identified by standard methods in the art, andin particular embodiments the antagonist is employed for treatment andor prevention of an individual in need thereof. In an additionalembodiment, the antagonist is employed in conjunction with an additionalcompound, such as a composition that modulates the NC_(Ca-ATP) channelof the invention. For example, the antagonist may be used in combinationwith an inhibitor of the channel of the invention. When employed incombination, the antagonist of a transcription factor of a SUR1-relatedpathway may be administered prior to, during, and/or subsequent to theadditional compound.

4. Antisense and Ribozymes

An antisense molecule that binds to a translational or transcriptionalstart site, or splice junctions, are ideal inhibitors. Antisense,ribozyme, and double-stranded RNA molecules target a particular sequenceto achieve a reduction or elimination of a particular polypeptide, suchas SUR1. Thus, it is contemplated that antisense, ribozyme, anddouble-stranded RNA, and RNA interference molecules are constructed andused to modulate SUR1 expression.

5. Antisense Molecules

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with complementary sequences. By complementary, it is meantthat polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, are employed to inhibit gene transcription or translation or bothwithin a host cell, either in vitro or in vivo, such as within a hostanimal, including a human subject.

constructs are designed to bind to the promoter and other controlregions, exons, introns or even exon-intron boundaries of a gene. It iscontemplated that the most effective antisense constructs may includeregions complementary to intron/exon splice junctions. Thus, antisenseconstructs with complementarity to regions within 50-200 bases of anintron-exon splice junction are used. It has been observed that someexon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

It is advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

6. RNA Interference

It is also contemplated in the present invention that double-strandedRNA is used as an interference molecule, e.g., RNA interference (RNAi).RNA interference is used to “knock down” or inhibit a particular gene ofinterest by simply injecting, bathing or feeding to the organism ofinterest the double-stranded RNA molecule. This technique selectively“knock downs” gene function without requiring transfection orrecombinant techniques (Giet, 2001; Hammond, 2001; Stein P, et al.,2002; Svoboda P, et al., 2001; Svoboda P, et al., 2000).

Another type of RNAi is often referred to as small interfering RNA(siRNA), which may also be utilized to inhibit SUR1. A siRNA maycomprises a double stranded structure or a single stranded structure,the sequence of which is “substantially identical” to at least a portionof the target gene (See WO 04/046320, which is incorporated herein byreference in its entirety). “Identity,” as known in the art, is therelationship between two or more polynucleotide (or polypeptide)sequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match of the order ofnucleotides between such sequences. Identity can be readily calculated.See, for example: Computational Molecular Biology, Lesk, A. M., ed.Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ea., Academic Press, New York, 1993, andthe methods disclosed in WO 99/32619, WO 01/68836, WO 00/44914, and WO01/36646, specifically incorporated herein by reference. While a numberof methods exist for measuring identity between two nucleotidesequences, the term is well known in the art. Methods for determiningidentity are typically designed to produce the greatest degree ofmatching of nucleotide sequence and are also typically embodied incomputer programs. Such programs are readily available to those in therelevant art. For example, the GCG program package (Devereux et al.),BLASTP, BLASTN, and FASTA (Atschul et al.,) and CLUSTAL (Higgins et al.,1992; Thompson, et al., 1994).

Thus, siRNA contains a nucleotide sequence that is essentially identicalto at least a portion of the target gene, for example, SUR1, or anyother molecular entity associated with the NC_(Ca-ATP) channel such asthe pore-forming subunit. One of skill in the art is aware that thenucleic acid sequences for SUR1 are readily available in GenBank, forexample, GenBank accession L40624, which is incorporated herein byreference in its entirety. Preferably, the siRNA contains a nucleotidesequence that is completely identical to at least a portion of thetarget gene. Of course, when comparing an RNA sequence to a DNAsequence, an “identical” RNA sequence will contain ribonucleotides wherethe DNA sequence contains deoxyribonucleotides, and further that the RNAsequence will typically contain a uracil at positions where the DNAsequence contains thymidine.

One of skill in the art will appreciate that two polynucleotides ofdifferent lengths may be compared over the entire length of the longerfragment. Alternatively, small regions may be compared. Normallysequences of the same length are compared for a final estimation oftheir utility in the practice of the present invention. It is preferredthat there be 100% sequence identity between the dsRNA for use as siRNAand at least 15 contiguous nucleotides of the target gene (e.g., SUR1),although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater mayalso be used in the present invention. A siRNA that is essentiallyidentical to a least a portion of the target gene may also be a dsRNAwherein one of the two complementary strands (or, in the case of aself-complementary RNA, one of the two self-complementary portions) iseither identical to the sequence of that portion or the target gene orcontains one or more insertions, deletions or single point mutationsrelative to the nucleotide sequence of that portion of the target gene.siRNA technology thus has the property of being able to toleratesequence variations that might be expected to result from geneticmutation, strain polymorphism, or evolutionary divergence.

There are several methods for preparing siRNA, such as chemicalsynthesis, in vitro transcription, siRNA expression vectors, and PCRexpression cassettes. Irrespective of which method one uses, the firststep in designing an siRNA molecule is to choose the siRNA target site,which can be any site in the target gene. In certain embodiments, one ofskill in the art may manually select the target selecting region of thegene, which may be an ORF (open reading frame) as the target selectingregion and may preferably be 50-100 nucleotides downstream of the “ATG”start codon. However, there are several readily available programsavailable to assist with the design of siRNA molecules, for examplesiRNA Target Designer by Promega, siRNA Target Finder by GenScriptCorp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA algorithm,siRNA program by Qiagen, Ambion siRNA predictor, Ambion siRNA predictor,Whitehead siRNA prediction, and Sfold. Thus, it is envisioned that anyof the above programs may be utilized to produce siRNA molecules thatcan be used in the present invention.

7. Ribozymes

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Forster and Symons,1987). For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., 1981; Reinhold-Hurek and Shub, 1992). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequencespecific cleavage/ligation reactions involving nucleic acids (Joyce,1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 reportsthat certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression is particularly suitedto therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990;Sioud et al., 1992). Most of this work involved the modification of atarget mRNA, based on a specific mutant codon that is cleaved by aspecific ribozyme. In light of the information included herein and theknowledge of one of ordinary skill in the art, the preparation and useof additional ribozymes that are specifically targeted to a given genewill now be straightforward.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992; Yuan and Altman, 1994), hairpinribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993)and hepatitis d virus based ribozymes (Perrotta and Been, 1992). Thegeneral design and optimization of ribozyme directed RNA cleavageactivity has been discussed in detail (Haseloff and Gerlach, 1988;Symons, 1992; Chowrira, et al., 1994; and Thompson, et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozymes,the cleavage site is a dinucleotide sequence on the target RNA, uracil(U) followed by either an adenine, cytosine or uracil (A,C or U;Perriman, et al., 1992; Thompson, et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al. (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in SUR1 targeted ribozymes is simply a matter of preparing andtesting a given sequence, and is a routinely practiced screening methodknown to those of skill in the art.

8. Inhibition of Post-Translational Assembly and Trafficking

Following expression of individual regulatory and pore-forming subunitproteins of the channel, and in particular aspects of the invention,these proteins are modified by glycosylation in the Golgi apparatus ofthe cell, assembled into functional heteromultimers that comprise thechannel, and then transported to the plasmalemmal membrane where theyare inserted to form functional channels. The last of these processes isreferred to as “trafficking”.

In specific embodiments of the invention, molecules that bind to any ofthe constituent proteins interfere with post-translational assembly andtrafficking, and thereby interfere with expression of functionalchannels. One such example is with glibenclamide binding to SUR1subunits. In additional embodiments, glibenclamide, which binds withfemtomolar affinity to SUR1, interferes with post-translational assemblyand trafficking required for functional channel expression.

B. Cation Channel Blockers

In some embodiments of the present invention, the combinatorialtherapeutic composition comprises one or more cation channel blockers(including, for example, TRPM4 blockers, Ca²⁺ channel blocker, K⁺channel blocker, Na⁺ channel blocker, and non-specific cation channelblocker). Exemplary TRPM4 blockers include pinokalant (LOE 908 MS);rimonabant (SR141716A); fenamates (flufenamic acid, mefenamic acid,niflumic acid, for example); SKF 96365(1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazolehydrochloride); and/or a combination or mixture thereof.

In certain embodiments a Ca2+ channel blocker includes, for example,Amlodipine besylate, (R)-(+)-Bay K, Cilnidipine, w-Conotoxin GVIA,w-Conotoxin MVIIC, Diltiazem hydrochloride, Gabapentin, Isradipine,Loperamide hydrochloride, Mibefradil dihydrochloride, Nifedipine,(R)-(−)-Niguldipine hydrochloride, (S)-(+)-Niguldipine hydrochloride,Nimodipine, Nitrendipine, NNC 55-0396 dihydrochloride, Ruthenium Red,SKF 96365 hydrochloride, SR 33805 oxalate, Verapamil hydrochloride.

In certain embodiments a K+ channel blocker includes, for example,Apamin, Charybdotoxin, Dequalinium dichloride, Iberiotoxin, Paxilline,UCL 1684, Tertiapin-Q, AM 92016 hydrochloride, Chromanol 293B,(−)-[3R,4S]-Chromanol 293B, CP 339818 hydrochloride, DPO-1, E-4031dihydrochloride, KN-93, Linopirdine dihydrochloride, XE 991dihydrochloride, 4-Aminopyridine, DMP 543, YS-035 hydrochloride.

In certain embodiments a Na+ channel blocker includes, for example,Ambroxol hydrochloride, Amiloride hydrochloride, Flecainide acetate,Flunarizine dihydrochloride, Mexiletine hydrochloride, QX 222, QX 314bromide, QX 314 chloride, Riluzole hydrochloride, Tetrodotoxin,Vinpocetine.

In certain embodiments a non-specific cation channel blocker includes,for example, Lamotrigine or Zonisamide.

In other embodiments of the present invention, the combinatorialtherapeutic composition comprises one or more glutamate receptorblockers including, for example, D-AP5, DL-AP5, L-AP5, D-AP7, DL-AP7,(R)-4-Carboxyphenylglycine, CGP 37849, CGP 39551, CGS 19755,(2R,3S)-Chlorpheg, Co 101244 hydrochloride, (R)-CPP, (RS)-CPP,D-CPP-ene, LY 235959, PMPA, PPDA, PPPA, Ro 04-5595 hydrochloride, Ro25-6981 maleate, SDZ 220-040, SDZ 220-581,(±)-1-(1,2-Diphenylethyl)piperidine maleate, IEM 1460, Loperamidehydrochloride, Memantine hydrochloride, (−)-MK 801 maleate, (+)-MK 801maleate, N20C hydrochloride, Norketamine hydrochloride, Remacemidehydrochloride, ACBC, CGP 78608 hydrochloride, 7-Chlorokynurenic acid,CNQX, 5,7-Dichlorokynurenic acid, Felbamate, Gavestinel, (S)-(−)-HA-966,L-689,560, L-701,252, L-701,324, Arcaine sulfate, Eliprodil,N-(4-Hydroxyphenylacetyl)spermine, N-(4-Hydroxyphenylpropanoyl) sperminetrihydrochloride, Ifenprodil hemitartrate, Synthalin sulfate, CFM-2,GYKI 52466 hydrochloride, IEM 1460, ZK 200775, NS 3763, UBP 296, UBP301, UBP 302, CNQX, DNQX, Evans Blue tetrasodium salt, NBQX, SYM 2206,UBP 282, and ZK 200775.

C. Antagonists of Specific Molecules

Antagonists of specific molecules may be employed, for example, thoserelated to endothelial dysfunction.

1. Antagonists of VEGF

Antagonists of VEGF may be employed. The antagonists may be synthetic ornatural, and they may antagonize directly or indirectly. VEGF TrapR1R2(Regeneron Pharmaceuticals, Inc.); Undersulfated, low-molecular-weightglycol-split heparin (Pisano et al., 2005); soluble NRP-1 (sNRP-1);Avastin (Bevacizumab); HuMV833; s-Fit-1, s-Flk-1; s-Flt-1/Flk-1; NM-3;and/or GFB 116.

2. Antagonists of MMP

Antagonists of any MMP may be employed. The antagonists may be syntheticor natural, and they may antagonize directly or indirectly. Exemplaryantagonists of MMPs include at least(2R)-2-[(4-biphenylsulfonyl)amino]-3-phenylproprionic acid (compound5a), an organic inhibitor of MMP-2/MMP-9 (Nyormoi et al., 2003);broad-spectrum MMP antagonist GM-6001 (Galardy et al., 1994; Graesser etal., 1998); TIMP-1 and/or TIMP-2 (Rolli et al., 2003); hydroxamate-basedmatrix metalloproteinase inhibitor (RS 132908) (Moore et al., 1999);batimastat (Corbel et al., 2001); those identified in United StatesApplication 20060177448 (which is incorporated by reference herein inits entirety); and/or marimastat (Millar et al., 1998); peptideinhibitors that comprise HWGF (including CTTHWGFTLC; SEQ ID NO:15)(Koivunen et al., 1999); and combinations thereof.

3. Antagonists of NOS

Antagonists of NOS may be employed. The antagonists may be synthetic ornatural, and they may antagonize directly or indirectly. The antagonistsmay be antagonists of NOS I, NOS II, NOS III, or may be nonselective NOSantagonists. Exemplary antagonists include at least the following:aminoguanidine (AG); 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT);S-ethylisothiourea (EIT) (Rairigh et al., 1998); asymmetricdimethylarginine (ADMA) (Vallance et al., 1992); N-nitro-L-argininemethylester (L-NAME) (Papapetropoulos et al., 1997; Babaei et al.,1998); nitro-L-arginine (L-NA) (Abman et al., 1990; Abman et al., 1991;Cornfield et al., 1992; Fineman et al., 1994; McQueston et al., 1993;Storme et al., 1999); the exemplary selective NOS II antagonists,aminoguanidine (AG) and N-(3-aminomethyl) benzylacetamidinedihydrochloride (1400W); NG-monomethyl-L-arginine (L-NMMA); theexemplary selective NOS I antagonist, 7-nitroindazole (7-NINA), and anonselective NOS antagonist, N-nitro-L-arginine (L-NNA), or a mixture orcombination thereof.

4. Antagonists of Thrombin

Antagonists of thrombin may be employed. The antagonists may besynthetic or natural, and they may antagonize directly or indirectly.Exemplary thrombin antagonists include at least the following:ivalirudin (Kleiman et al., 2002); hirudin (Hoffman et al., 2000);SSR182289 (Duplantier et al., 2004); antithrombin III; thrombomodulin;Lepirudin (Refludan, a recombinant therapeutic hirudin); P-PACK II(d-Phenylalanyl-L-Phenylalanylarginine-chloro-methyl ketone 2 HCl);Thromstop® (BNas-Gly-(pAM)Phe-Pip); Argatroban (Carr et al., 2003); andmixtures or combinations thereof.

D. Others

Non-limiting examples of an additional pharmacological therapeutic agentthat may be used in the present invention include anantihyperlipoproteinemic agent, an antiarteriosclerotic agent, ananticholesterol agent, an antiinflammatory agent, anantithrombotic/fibrinolytic agent, antiplatelet, vasodilator, and/ordiuretics. Anticholesterol agents include but are not limited to HMG-CoAReductase inhibitors, cholesterol absorption inhibitors, bile acidsequestrants, nicotinic acid and derivatives thereof, fibric acid andderivatives thereof. HMG-CoA Reductase inhibitors include statins, forexample, but not limited to atorvastatin calcium (Lipitor®),cerivastatin sodium (Baycol®), fluvastatin sodium (Lescol®), lovastatin(Advicor®), pravastatin sodium (Pravachol®), and simvastatin (Zocor®).Agents known to reduce the absorption of ingested cholesterol include,for example, Zetia®. Bile acid sequestrants include, but are not limitedto cholestyramine, cholestipol and colesevelam. Other anticholesterolagents include fibric acids and derivatives thereof (e.g., gemfibrozil,fenofibrate and clofibrate); nicotinic acids and derivatives thereof(e.g., nician, lovastatin) and agents that extend the release ofnicotinic acid, for example niaspan. Antiinflammatory agents include,but are not limited to non-steroidal anti-inflammatory agents (e.g.,naproxen, ibuprofen, celeoxib) and steroidal anti-inflammatory agents(e.g., glucocorticoids). Diuretics include, but are not limited to suchas furosemide (Lasix®), bumetanide (Bumex®), torsemide (Demadex®),thiazide & thiazide-like diuretics (e.g., chlorothiazide (Diuril®) andhydrochlorothiazide (Esidrix®), benzthiazide, cyclothiazide, indapamide,chlorthalidone, bendroflumethizide, metolazone), amiloride, triamterene,and spironolacton. Vasodilators include, but are not limited tonitroglycerin.

In only certain embodiments that would not be contraindicated forco-administration with an inhibitor of the NC_(Ca-ATP) channel,additional pharmacological therapeutic agents includeantithrombotic/fibrinolytic agent, anticoagulant, antiplatelet,vasodilator, and/or diuretics. Thromoblytics that are used can include,but are not limited to prourokinase, streptokinase, and tissueplasminogen activator (tPA). Anticoagulants include, but are not limitedto heparin, warfarin, and coumadin. Antiplatelets include, but are notlimited to aspirin, and aspirin related-compounds, for exampleacetaminophen. Thus, in certain embodiments, the present inventioncomprises co-administration of an antagonist of the NC_(Ca-ATP) channelwith a thrombolytic agent. Co-administration of these two compounds willincrease the therapeutic window of the thrombolytic agent. Examples ofsuitable thrombolytic agents that can be employed in the methods andpharmaceutical compositions of this invention are prourokinase,streptokinase, and tissue plasminogen activator (tPA).

In certain embodiments, the present invention comprisesco-administration of an antagonist of the NC_(Ca-ATP) channel withglucose or related carbohydrate to maintain appropriate levels of serumglucose. Appropriate levels of blood glucose are within the range ofabout 60 mmol/l to about 150 mmol/liter. Thus, glucose or a relatedcarbohydrate is administered in combination to maintain the serumglucose within this range.

VI. Exemplary Pharmaceutical Formulations and Methods of Use

In particular embodiments, the invention employs pharmaceuticalformulations comprising a singular or combinatorial composition thatinhibits a NC_(Ca-ATP) channel.

A. Exemplary Compositions of the Present Invention

The present invention also contemplates therapeutic methods employingcompositions comprising the active substances disclosed herein.Preferably, these compositions include pharmaceutical compositionscomprising a therapeutically effective amount of one or more of theactive compounds or substances along with a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically acceptable” carrier means anon-toxic, inert solid, semi-solid liquid filler, diluent, encapsulatingmaterial, formulation auxiliary of any type, or simply a sterile aqueousmedium, such as saline. Some examples of the materials that can serve aspharmaceutically acceptable carriers are sugars, such as lactose,glucose and sucrose, starches such as corn starch and potato starch,cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt,gelatin, talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol,polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate and ethyl laurate, agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. Examples ofpharmaceutically acceptable antioxidants include, but are not limitedto, water soluble antioxidants such as ascorbic acid, cysteinehydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite,and the like; oil soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, aloha-tocopherol and the like; and the metalchelating agents such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

B. Dose Determinations

By a “therapeutically effective amount” or simply “effective amount” ofan active compound, such as glibenclamide or tolbutamide, for example,is meant a sufficient amount of the compound to treat or alleviate thespinal cord injury at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the active compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular patient will depend upon a variety of factors includingthe disorder being treated and the severity of the spinal cord injury;activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coinciding with the specificcompound employed; and like factors well known in the medical arts.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell assays or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell based assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by high performance liquid chromatography.

The total daily dose of the active compounds of the present inventionadministered to a subject in single or in divided doses can be inamounts, for example, from 0.01 to 25 mg/kg body weight or more usuallyfrom 0.1 to 15 mg/kg body weight. Single dose compositions may containsuch amounts or submultiples thereof to make up the daily dose. Ingeneral, treatment regimens according to the present invention compriseadministration to a human or other mammal in need of such treatment fromabout 1 mg to about 1000 mg of the active substance(s) of this inventionper day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg,100 mg, 500 mg or 1000 mg.

In certain situations, it may be important to maintain a fairly highdose of the active agent in the blood stream of the patient,particularly early in the treatment. Such a fairly high dose may includea dose that is several times greater than its use in other indications.For example, the typical anti-diabetic dose of oral or IV glibenclamideis about 2.5 mg/kg to about 15 mg/kg per day; the typical anti-diabeticdose of oral or IV tolbutamide is about to 0.5 gm/kg to about 2.0 gm/kgper day; the typical anti-diabetic dose for oral gliclazide is about 30mg/kg to about 120 mg/kg per day; however, much larger doses may berequired to block spinal cord damage and/or PHN.

For example, in one embodiment of the present invention directed to amethod of preventing neuronal cell swelling in the brain of a subject byadministering to the subject a formulation containing an effectiveamount of a compound that blocks the NC_(Ca-ATP) channel and apharmaceutically acceptable carrier; such formulations may contain fromabout 0.1 to about 100 grams of tolbutamide or from about 0.5 to about150 milligrams of glibenclamide. In another embodiment of the presentinvention directed to a method of alleviating the negative effects oftraumatic brain injury or cerebral ischemia stemming from neural cellswelling in a subject by administering to the subject a formulationcontaining an effective amount of a compound that blocks the NC_(Ca-ATP)channel and a pharmaceutically acceptable carrier.

In situations of spinal cord injury and/or PHN, it may be important tomaintain a fairly high dose of the active agent to ensure delivery tothe brain of the patient, particularly early in the treatment. Hence, atleast initially, it may be important to keep the dose relatively highand/or at a substantially constant level for a given period of time,preferably, at least about six or more hours, more preferably, at leastabout twelve or more hours and, most preferably, at least abouttwenty-four or more hours. In situations of traumatic brain injury orcerebral ischemia (such as stroke), it may be important to maintain afairly high dose of the active agent to ensure delivery to the brain ofthe patient, particularly early in the treatment.

C. Formulations and Administration

The compounds of the present invention may be administered alone or incombination or in concurrent therapy with other agents which affect thecentral or peripheral nervous system.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs containing inert diluents commonly used in the art, such aswater, isotonic solutions, or saline. Such compositions may alsocomprise adjuvants, such as wetting agents; emulsifying and suspendingagents; sweetening, flavoring and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulation can be sterilized, for example, by filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions, which can be dissolvedor dispersed in sterile water or other sterile injectable medium justprior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of a drug from subcutaneous or intramuscular injection.The most common way to accomplish this is to inject a suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug becomes dependent on the rate of dissolutionof the drug, which is, in turn, dependent on the physical state of thedrug, for example, the crystal size and the crystalline form. Anotherapproach to delaying absorption of a drug is to administer the drug as asolution or suspension in oil. Injectable depot forms can also be madeby forming microcapsule matrices of drugs and biodegradable polymers,such as polylactide-polyglycoside. Depending on the ratio of drug topolymer and the composition of the polymer, the rate of drug release canbe controlled. Examples of other biodegradable polymers includepolyorthoesters and polyanhydrides. The depot injectables can also bemade by entrapping the drug in liposomes or microemulsions, which arecompatible with body tissues.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable non-irritating excipient, such as cocoabutter and polyethylene glycol which are solid at ordinary temperaturebut liquid at the rectal temperature and will, therefore, melt in therectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, gelcaps and granules. In such solid dosageforms the active compound may be admixed with at least one inert diluentsuch as sucrose, lactose or starch. Such dosage forms may also comprise,as is normal practice, additional substances other than inert diluents,e.g., tableting lubricants and other tableting aids such as magnesiumstearate and microcrystalline cellulose. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings andother release-controlling coatings.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings and other coatings well known in thepharmaceutical formulating art. They may optionally contain opacifyingagents and can also be of a composition that they release the activeingredient(s) only, or preferably, in a certain part of the intestinaltract, optionally in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention further include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. Transdermal patcheshave the added advantage of providing controlled delivery of activecompound to the body. Such dosage forms can be made by dissolving ordispersing the compound in the proper medium. Absorption enhancers canalso be used to increase the flux of the compound across the skin. Therate can be controlled by either providing a rate controlling membraneor by dispersing the compound in a polymer matrix or gel. The ointments,pastes, creams and gels may contain, in addition to an active compoundof this invention, excipients such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

The method of the present invention employs the compounds identifiedherein for both in vitro and in vivo applications. For in vivoapplications, the invention compounds can be incorporated into apharmaceutically acceptable formulation for administration. Those ofskill in the art can readily determine suitable dosage levels when theinvention compounds are so used.

As employed herein, the phrase “suitable dosage levels” refers to levelsof compound sufficient to provide circulating concentrations high enoughto effectively block the NC_(Ca-ATP) channel and prevent or reducespinal cord injury and/or PHN.

In accordance with a particular embodiment of the present invention,compositions comprising at least one SUR1 antagonist compound (asdescribed above), and a pharmaceutically acceptable carrier arecontemplated.

In accordance with a particular embodiment of the present invention,compositions comprising at least one TRPM4 antagonist compound (asdescribed above), and a pharmaceutically acceptable carrier arecontemplated.

In accordance with a particular embodiment of the present invention,compositions comprising a combination of at least one SUR1 antagonistcompound and at least one TRPM4 antagonist compound (as describedabove), and a pharmaceutically acceptable carrier are contemplated.

Exemplary pharmaceutically acceptable carriers include carriers suitablefor oral, intravenous, subcutaneous, intramuscular, intracutaneous, andthe like administration. Administration in the form of creams, lotions,tablets, dispersible powders, granules, syrups, elixirs, sterile aqueousor non-aqueous solutions, suspensions or emulsions, and the like, iscontemplated.

For the preparation of oral liquids, suitable carriers includeemulsions, solutions, suspensions, syrups, and the like, optionallycontaining additives such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitablecarriers include sterile aqueous or non-aqueous solutions, suspensions,or emulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized, for example,by filtration through a bacteria-retaining filter, by incorporatingsterilizing agents into the compositions, by irradiating thecompositions, or by heating the compositions. They can also bemanufactured in the form of sterile water, or some other sterileinjectable medium immediately before use. The active compound is admixedunder sterile conditions with a pharmaceutically acceptable carrier andany needed preservatives or buffers as may be required.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition (anantagonist of the NC_(Ca-ATP) channel or its related-compounds thereof)calculated to produce the desired responses in association with itsadministration, e.g., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime.

D. Formulations and Routes for Administration of Compounds

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more modulators of NC_(Ca-ATP) channel(antagonist) or related-compounds or additional agent dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one modulators of NC_(Ca-ATP) channel(antagonist) or related-compounds or additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The modulators of NC_(Ca-ATP) channel (antagonist) or related-compoundsmay comprise different types of carriers depending on whether it is tobe administered in solid, liquid or aerosol form, and whether it need tobe sterile for such routes of administration as injection. The presentinvention can be administered intravenously, intradermally,transdermally, intrathecally, intraventricularly, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The modulators of NC_(Ca-ATP) channel (antagonist) or related-compoundsmay be formulated into a composition in a free base, neutral or saltform. Pharmaceutically acceptable salts, include the acid additionsalts, e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentarily administrations such asdrug release capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include modulators ofNC_(Ca-ATP) channel (antagonist) or related-compounds, one or morelipids, and an aqueous solvent. As used herein, the term “lipid” will bedefined to include any of a broad range of substances that ischaracteristically insoluble in water and extractable with an organicsolvent. This broad class of compounds is well known to those of skillin the art, and as the term “lipid” is used herein, it is not limited toany particular structure. Examples include compounds which containlong-chain aliphatic hydrocarbons and their derivatives. A lipid may benaturally occurring or synthetic (i.e., designed or produced by man).However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the modulators of NC_(Ca-ATP) channel (antagonist)or related-compounds may be dispersed in a solution containing a lipid,dissolved with a lipid, emulsified with a lipid, mixed with a lipid,combined with a lipid, covalently bonded to a lipid, contained as asuspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure by anymeans known to those of ordinary skill in the art. The dispersion may ormay not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeutic and/orprophylatic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according tot he response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

Pharmaceutical formulations may be administered by any suitable route ormeans, including alimentarily, parenteral, topical, mucosal or otherroute or means of administration. Alimentarily routes of administrationinclude administration oral, buccal, rectal and sublingual routes.Parenteral routes of administration include administration includeinjection into the brain parenchyma, and intravenous, intradermal,intramuscular, intraarterial, intrathecal, subcutaneous,intraperitoneal, and intraventricular routes of administration. Topicalroutes of administration include transdermal administration.

E. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the modulators ofNC_(Ca-ATP) channel (antagonist) or related-compounds are formulated tobe administered via an alimentarily route. Alimentarily routes includeall possible routes of administration in which the composition is indirect contact with the alimentarily tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes ofalimentarily administration include suppositories. Suppositories aresolid dosage forms of various weights and shapes, usually medicated, forinsertion into the rectum. After insertion, suppositories soften, meltor dissolve in the cavity fluids. In general, for suppositories,traditional carriers may include, for example, polyalkylene glycols,triglycerides or combinations thereof. In certain embodiments,suppositories may be formed from mixtures containing, for example, theactive ingredient in the range of about 0.5% to about 10%, andpreferably about 1% to about 2%.

F. Parenteral Compositions and Formulations

In further embodiments, modulators of NC_(Ca-ATP) channel (antagonist)or related-compounds may be administered via a parenteral route. As usedherein, the term “parenteral” includes routes that bypass thealimentarily tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered for example, but not limited tointravenously, intradermally, intramuscularly, intraarterially,intraventricularly, intrathecally, subcutaneous, or intraperitoneallyU.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515;and 5,399,363 (each specifically incorporated herein by reference in itsentirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, DMSO, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

G. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundmodulators of NC_(Ca-ATP) channel (antagonist) or related-compounds maybe formulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

VII. Kits of the Invention

Any of the compositions described herein may be comprised in a kit, andthe kit may be employed for therapeutic and/or preventative purposes,including for IVH, SCI, and/or PHN. Antagonists of the channel(regulatory subunit or pore-forming) that may be provided include butare not limited to sulfonylurea compounds, benzamido derivatives,antibodies (monoclonal or polyclonal, for example to SUR1 or TRPM4),SUR1 oligonucleotides, SUR1 polypeptides, TRPM4 oligonucleotides, TRPM4polypeptides, small molecules or combinations thereof, antagonist, etc.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one components in the kit, the kitalso may generally contain a second, third or other additional containerinto which additional components may be separately placed. However,various combinations of components may be comprised in a vial. The kitsof the present invention also will typically include a means forcontaining the SUR1 inhibitor, lipid, additional agent, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow molded plastic containers intowhich the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The SUR1 antagonist orrelated-compounds thereof may also be formulated into a syringeablecomposition. In which case, the container means may itself be a syringe,pipette, and/or other such like apparatus, from which the formulationmay be applied to an infected area of the body, injected into an animal,and/or even applied to and/or mixed with the other components of thekit. Examples of aqueous solutions include, but are not limited toethanol, DMSO and/or Ringer's solution. In certain embodiments, theconcentration of DMSO or ethanol that is used is no greater than 0.1% or(1 ml/1000 L).

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which the SUR1antagonist or related-compounds thereof is suitably allocated. The kitsmay also comprise a second container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of thecomposition(s) of the invention within the body of an animal. Such aninstrument may be a syringe, pipette, forceps, and/or any such medicallyapproved delivery vehicle.

In addition to the SUR1 antagonist or related-compounds thereof, thekits may also include a second active ingredient. Examples of the secondactive ingredient include substances to prevent hypoglycemia (e.g.,glucose, D5W, glucagon, etc.), statins, diuretics, vasodilators, etc.These second active ingredients may be combined in the same vial as theSUR1 antagonist or related-compounds thereof or they may be contained ina separate vial. In a specific embodiment, a combinatorial therapeuticcomposition is provided in a kit, and in some embodiments the two ormore compounds that make up the composition are housed separately or asa mixture. Other second active ingredients may be employed so long asthey are not contra-indicated and would not worsen bleeding, forexample, such as thrombolytic agents, anticoagulants, and/orantiplatelets, for example.

Still further, the kits of the present invention can also includeglucose-testing kits. Thus, the blood glucose of the patient is measuredusing the glucose testing kit, then the SUR1 antagonist orrelated-compounds thereof can be administered to the subject followed bymeasuring the blood glucose of the patient.

In addition to the above kits, the kits of the present invention can beassembled such that an IV bag comprises a septum or chamber which can beopened or broken to release the compound into the IV bag. Another typeof kit may include a bolus kit in which the bolus kit comprises apre-loaded syringe or similar easy to use, rapidly administrable device.An infusion kit may comprise the vials or ampoules and an IV solution(e.g., Ringer's solution) for the vials or ampoules to be added prior toinfusion. The infusion kit may also comprise a bolus kit for abolus/loading dose to be administered to the subject prior, during orafter the infusion.

Any of the compositions described herein may be comprised in a kit. In aspecific embodiment, a combinatorial therapeutic composition is providedin a kit, and in some embodiments the two or more compounds that make upthe composition are housed separately or as a mixture. Antagonists ofthe channel that may be provided include but are not limited toantibodies (monoclonal or polyclonal), SUR1 oligonucleotides, SUR1polypeptides, small molecules or combinations thereof, antagonist, etc.

Therapeutic kits of the present invention are kits comprising anantagonist or an related-compound thereof. Depending upon the conditionand/or disease that is being treated, the kit may comprise an SUR1antagonist or related-compound thereof to block and/or inhibit theNC_(Ca-ATP) channel. The kit may comprise a TRPM4 antagonist orrelated-compound thereof to block and/or inhibit the NC_(Ca-ATP)channel. The kit may comprise both a TRPM4 antagonist orrelated-compound thereof and a SUR1 antagonist or related compoundthereof to block and/or inhibit the NC_(Ca-ATP) channel. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of SUR1 antagonist, TRPM4 antagonist, orrelated-compound thereof. The kit may have a single container means,and/or it may have distinct container means for each compound. Forexample, the therapeutic compound and solution may be contained withinthe same container; alternatively, the therapeutic compound and solutionmay each be contained within different containers. A kit may include acontainer with the therapeutic compound that is contained within acontainer of solution.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The SUR1 antagonist orrelated-compounds thereof may also be formulated into a syringeablecomposition. In which case, the container means may itself be a syringe,pipette, and/or other such like apparatus, from which the formulationmay be applied to an infected area of the body, injected into an animal,and/or even applied to and/or mixed with the other components of thekit.

Examples of aqueous solutions include, but are not limited to ethanol,DMSO and/or Ringer's solution. In certain embodiments, the concentrationof DMSO or ethanol that is used is no greater than 0.1% or (1 ml/1000L). However, the components of the kit may be provided as driedpowder(s). When reagents and/or components are provided as a dry powder,the powder can be reconstituted by the addition of a suitable solvent.It is envisioned that the solvent may also be provided in anothercontainer means.

VIII. Insurance Processing Embodiments

In one embodiment of the invention, there is provided a method forprocessing an insurance claim for diagnosis and/or treatment of amedical condition of the invention using a composition(s) of theinvention as disclosed herein and/or using a treatment method asdisclosed herein. In a specific embodiment, the method employs acomputer for said processing of an insurance claim. In further specificembodiments, the dosage for the composition may be any suitable dosagefor treatment of the medical condition.

In embodiments of the present invention, a subject, in particular ahuman subject, may be examined and/or may be diagnosed as sufferingfrom, or being at risk of, a disease or condition selected from, forexample, progressive hemorrhagic necrosis following spinal cord injury,traumatic brain injury, subarachnoid hemorrhage, and/or intraventricularhemorrhage. Such an examination may be performed by, for example, aphysician, including a general practice physician or a specialist, suchas an emergency room physician, a trauma specialist, an internist, aneurologist, a cardiologist, or other specialist; may be performed by anurse, physician's assistant, medic, ambulance attendant, or otherhealth professional. Examination and/or diagnosis may be performedanywhere, including at the scene of an accident or disaster; in anambulance or other medial transport vehicle; in a clinic; in anexamining room; in a hospital, including in any room or part of ahospital; in an extended care facility; or other health care facility.Such an examination may be an emergency examination, and/or aperfunctory examination, and or a minimally detailed examination, or maybe an extended and detailed examination.

Such an examination may be performed without the use of clinicalequipment or devices, or with some use of clinical equipment anddevices, and may include the use of sophisticated clinical and/ordiagnostic equipment and/or devices, which may include, for example,computer assisted tomography, magnetic resonance imaging, positronemission tomography, X-ray, ultrasound, or other imaging equipment;angiography, or other invasive procedures; and other medical equipmentand procedures.

Such a diagnosis may be made as a result of an examination as discussedabove, or may be made in the absence of an examination.

A medical practitioner, nurse, clinical or emergency technician or otherperson may provide medical assistance and diagnostic assistance in thecourse of providing routine, elective, or emergency medical care. In anycase, all or part of the cost of such care, such procedures, suchdiagnostic work, and such diagnoses may be reimbursed by an insuranceplan, employment agreement, government program, or other arrangementfrom which the subject may benefit. For example, a human subject may becovered by an insurance policy which pays for and/or reimburses(“covers”) medical costs incurred by the subject.

As disclosed herein, a method for processing an insurance claim fordiagnosis and/or treatment of a medical condition of the invention asdisclosed herein, for a subject who has received medical treatment forprogressive hemorrhagic necrosis following spinal cord injury, traumaticbrain injury, subarachnoid hemorrhage, and/or intraventricularhemorrhage, includes the steps of:

i) receiving a claim for a medical treatment, procedure, and/ormedicament for treating for progressive hemorrhagic necrosis followingspinal cord injury, traumatic brain injury, subarachnoid hemorrhage,and/or intraventricular hemorrhage with a SUR1 antagonist, a TRPM4antagonist, or combination thereof; and

ii) providing reimbursement for the medical treatment, procedure, and/ormedicament.

In a further embodiment, a method for processing an insurance claim fordiagnosis and/or treatment of a medical condition of the invention asdisclosed herein, for a subject who has received medical treatment forprogressive hemorrhagic necrosis following spinal cord injury, traumaticbrain injury, subarachnoid hemorrhage, and/or intraventricularhemorrhage, includes the steps of:

i) receiving a claim for a medical treatment, procedure, and/ormedicament for treating for progressive hemorrhagic necrosis followingspinal cord injury, traumatic brain injury, subarachnoid hemorrhage,and/or intraventricular hemorrhage with a SUR1 antagonist, a TRPM4antagonist, or both;

ii) evaluating the claim for a medical treatment, procedure, and/ormedicament; and

ii) providing reimbursement for the medical treatment, procedure, and/ormedicament.

In embodiments of these methods for processing an insurance claim, anyone or more of the steps may involve the use of a computer; any one ormore of the steps may involve the use of electronic data transfer; anyone or more of the steps may involve the use of a telephone and/orfacsimile device; any one or more of the steps may involve the use ofmail and/or of a delivery service; and any one or more of the steps mayinvolve the use of electronic fund transfer devices and/or methods.

In embodiments of these methods for processing an insurance claim, thetreatment may include a treatment or medicament comprising any suitabledosage of a SUR1 antagonist, a TRPM4 antagonist, or combination thereof,for treatment of the medical condition.

In particular embodiments of the methods for processing an insuranceclaim, the treatment and/or medicament is directed to, or affects, theNC_(Ca-ATP) channel.

In particular embodiments of the methods for processing an insuranceclaim, the treatment and/or medicament uses or includes a SUR1antagonist such as, for example, glibenclamide and tolbutamide,repaglinide, nateglinide, meglitinide, midaglizole, LY397364, LY389382,glyclazide, glimepiride, estrogen, estrogen related-compounds(estradiol, estrone, estriol, genistein, non-steroidal estrogen (e.g.,diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone,etc.), and compounds known to inhibit or block KATP channels.

In particular embodiments of the methods for processing an insuranceclaim, the treatment and/or medicament uses or includes a TRPM4antagonist such as, for example, flufenamic acid, pinkolant, rimonabant,or a fenamate (such as flufenamic acid, mefenamic acid, meclofenamicacid, or niflumic acid),1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazolehydrochloride, and a biologically active derivative thereof.

In particular embodiments of the methods for processing an insuranceclaim, the treatment and/or medicament uses or includes a SUR1antagonist and a TRPM4 antagonist.

In further embodiments of the methods for processing an insurance claim,the treatment and/or medicament uses or includes a treatment and/ormedicament is directed to, or affects, the NC_(Ca-ATP) channel, where atreatment and/or medicament is directed to, or affects, the NC_(Ca-ATP)channel includes or uses a non-sulfonyl urea compound, such as 2,3-butanedione and 5-hydroxydecanoic acid, quinine, and therapeuticallyequivalent salts and derivatives thereof; a protein, a peptide, anucleic acid (such as an RNAi molecule or antisense RNA, includingsiRNA), or a small molecule that antagonizes or reduces the activity ofthe NC_(Ca-ATP) channel; and/or includes or uses MgADP.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Up-Regulation of SUR1 in SCI

SUR1 expression was studied in spinal cords of uninjured rats and ratsafter “severe” SCI (10-gm weight dropped 25 mm; 3-5 rats/group)(Soblosky et al. 2001; Gensel et al., 2006). In controls, low levels ofSUR1 expression were found in the dorsal horns (FIG. 1A), due toconstitutively expressed KATP channels (Yamashita et al., 1994).

After unilateral SCI, the lesion itself as well as the pattern of SUR1expression changed with time and distance from the impact site (FIG.1A). Early post-SCI (¾ h), the lesion was small and was notimmunolabeled by anti-SUR1 antibody (not shown). At 6 h, a necroticlesion was apparent as a void in the ipsilateral cord, and SUR1up-regulation was prominent in tissues surrounding the void. At 24 h,the necrotic lesion had enlarged (Nelson et al., 1977; Tator, 1995),SUR1 up-regulation was still apparent in the rim of the necrotic lesion,but now it extended to tissues more distant from the impact site,including into the contralateral hemi-cord. Immunolabeling for SUR2 wasdetected only in vascular smooth muscle cells of pial arterioles, bothpre- and post-SCI.

In the “core” of the lesion (heavily labeled area in FIG. 1A, 6 h), SUR1up-regulation was present in various cells and structures, includinglarge ballooned neuron-like cells and capillary-like elongatedstructures (FIG. 1B). In the “penumbra” (tissue adjacent to the heavilylabeled core in FIG. 1A, 6 h), SUR1 up-regulation was associatedpredominantly with capillaries (FIGS. 1C, 1D).

Up-regulation of SUR1 was confirmed with immunoblots. With the amount ofprotein loaded, SUR1 was not detectable in normal cords, whereas aprominent, single band at ˜190 kDa (Simard et al., 2006) was observed 6h post-SCI (FIG. 1E). The blood introduced into the tissues by theinjury did not account for the increase in SUR1 (FIG. 1E). In situhybridization confirmed widespread expression of SUR1 after injury,especially in capillaries and post-capillary venules in the penumbra(FIGS. 1F, 1G).

Example 2 SUR1 in Endothelium is Associated with NC_(Ca-ATP) Channel

SUR1 forms the regulatory subunit of both NC_(Ca-ATP) and some K_(ATP)channels (Chen et al., 2003). Our previous work demonstrated that,following exposure to hypoxia or ischemia in vivo, up-regulation of SUR1in astrocytes and neurons is associated with expression of functionalNC_(Ca-ATP) channels, not K_(ATP) channels (Chen et al., 2003, Simard etal., 2006). The same reports also showed up-regulation of SUR1 incapillaries, as was found here with SCI, but the associated channel wasnot identified. Endothelial cells may normally express K_(ATP) channels,but the regulatory subunit of cardiovascular K_(ATP) channels isgenerally SUR2, not SUR1 (Jansen-Olesen et al., 2005). Nevertheless, itwas important to determine which of the two channels, K_(ATP) orNC_(Ca-ATP), the newly expressed SUR1 was associated with in capillaryendothelium.

Endothelial cell cultures from 3 sources, human brain microvascular,human aorta, and murine brain microvascular, were used to assess SUR1expression and characterize channel properties following exposure tohypoxia, with the same results observed with all 3. Control culturesshowed little expression of SUR1, but exposure to hypoxia for 24 hresulted in significant up-regulation of SUR1 (FIG. 2A). Insulinomacells, which constitutively express SUR1-regulated K_(ATP) channels,showed no up-regulation of SUR1 when exposed to the same hypoxicconditions (FIG. 2A).

Patch clamp of endothelial cells was performed using anystatin-perforated patch technique, to maintain the metabolic integrityof the cells. The identity of the activated channel can be assessed bymeasurement of the “reversal potential”, the potential at which an ionchannel current reverses from inward to outward. With physiologicallyrelevant concentrations of ions intracellularly and extracellularly(high potassium inside, high sodium outside), the reversal potential canunambiguously distinguish between a K⁺ channel current such as K_(ATP),which reverses negative to −50 mV and a non-selective cation channelcurrent such as NC_(Ca-ATP), which reverses near 0 mV.

Channel activation by diazoxide was studied, which opens SUR-regulatedchannels without ATP depletion and, of SUR activators, is the mostselective for SUR1 over SUR2 (Chen et al., 2003). Patch clamp ofendothelial cells cultured under normoxic conditions showed thatdiazoxide either had no effect or, in half of the cells, activated anoutwardly rectifying current that reversed at potentials more negativethan −50 mV, consistent with a K_(ATP) channel (FIG. 2B) (Seino, 1999).By contrast, in most endothelial cells cultured under hypoxicconditions, diazoxide activated an ohmic current that reversed near 0 mVand that was inward at −50 mV (FIG. 2B), which is incompatible withK_(ATP), but consistent with NC_(Ca-ATP) channels (Chen and Simard,2001; Chen et al., 2003; Simard et al., 2006).

Channel activation induced by Na azide was also studied, which is amitochondrial uncoupler that depletes cellular ATP (Chen and Simard,2001). In most endothelial cells exposed to hypoxic conditions, Naazide-induced ATP depletion activated an ohmic current that was inwardat −50 mV, that reversed near 0 mV, and that was blocked by 1 μMglibenclamide (FIG. 2C), again consistent with NC_(Ca-ATP) channels.

Single channel recordings were performed using inside-out patches, withCs⁺ as the only permeant cation. This confirmed the presence of achannel that was sensitive to block by ATP on the cytoplasmic side andthat had a single channel conductance of 37 pS (FIG. 2D). These findingsare incompatible with K_(ATP) channels, which is not permeable to Cs⁺and which has a slope conductance of ˜75 pS, but are consistent withNC_(Ca-ATP) channels.

The characteristics of the channel identified in endothelial cells fromboth aorta and brain capillaries from 2 species, including expressiononly after exposure to hypoxia, activation by depletion of cellular ATPor diazoxide, a reversal potential near 0 mV, conductance of Cs⁺, andsingle channel conductance of 37 pS, reproduce exactly our previousfindings with NC_(Ca-ATP) channels in astrocytes and neurons (Chen andSimard, 2001; Chen et al., 2003; Simard et al., 2006), and reaffirm thatthe NC_(Ca-ATP) channel is not constitutively expressed, is up-regulatedonly with an appropriate insult, and when expressed, is inactive untilintracellular ATP is depleted.

Example 3 Glibenclamide Block of SUR1—Extravasation of Blood

To assess the role of SUR1 in SCI, the effect of glibenclamide wasstudied, which is a sulfonylurea inhibitor that binds with subnanomolaror nanomolar affinity (0.4-4.0 nM) to SUR1 (24). Immediately afterinjury, animals were implanted with mini-osmotic pumps that deliveredeither vehicle or glibenclamide (200 ng/h) s.q. Constant infusion of alow-dose of drug was used to achieve sustained occupancy of onlyhigh-affinity receptors.

Cords of vehicle-treated animals examined 24 h post-SCI showed prominentbleeding at the surface and internally, with internal bleedingconsisting of a central region of hemorrhage plus numerous distinctpetechial hemorrhages at the periphery (FIG. 3A, arrows). By contrast,cords of glibenclamide-treated animals showed visibly less hemorrhageand it was largely confined to the site of impact, with fewer petechialhemorrhages in surrounding tissues (FIG. 3A).

The amount of extravasated blood in tissue homogenates was quantified atdifferent times post-SCI, after first removing intravascular blood (FIG.3B). In cords from vehicle-treated animals, measurements showed aprogressive increase in the amount of blood, with a maximum reached ˜12h post-SCI (FIG. 3B). By contrast, cords from glibenclamide-treatedanimals showed little increase in extravasated blood during the 24 hafter injury, with most of the blood present at 24 h being attributableto the initial impact (FIG. 3B).

Formation of petechial hemorrhages implies catastrophic failure ofcapillary integrity. Capillaries in the region of injury were examinedby immunolabeling with vimentin, which is up-regulated in endotheliumfollowing injury (Haseloff et al., 2006). In controls, vimentin(+)capillaries appeared foreshortened or fragmented, whereas inglibenclamide-treated animals, the capillaries were elongated andappeared more normal (FIG. 3C).

In post-ischemic reperfusion of CNS tissues, catastrophic failure ofcapillary integrity has been attributed to the action of matrixmetalloproteinases (MMP) (Wang et al., 2004). It was assessed whetherglibenclamide might have an effect on MMP activity using zymography tomeasure gelatinase activity of recombinant MMP. Gelatinase activity wasnot affected by glibenclamide, although it was strongly inhibited by aspecific MMP inhibitor (FIG. 3D), indicating that the reduction inhemorrhage with glibenclamide could not be attributed to MMP inhibition.

Glibenclamide did not affect bleeding time (FIG. 3E), suggesting thatthe reduction in hemorrhage with glibenclamide following SCI wasunlikely to be due to an effect on coagulation or platelet function(Chan et al., 1982).

The dose of glibenclamide used resulted in a small decrease in serumglucose, from 236±15 to 201±20 (5 rats per group; p=0.19) measured 3 hafter SCI.

Example 4 Glibenclamide Block of SUR1—Lesion Size

Labeling of longitudinal sections for the astrocyte-marker, glialfibrillary acidic protein (GFAP) and for myelin revealed thatglibenclamide-treatment was associated with smaller lesions, lessreactive gliosis and better myelin preservation 24 h post-SCI comparedto controls (FIGS. 4A,4B). Similarly, hematoxylin and eosin staining ofcross sections showed that glibenclamide-treatment was associated withsmaller lesions 7 d post-SCI compared to controls (FIG. 4C). Invehicle-treated controls at both 1 and 7 d, the lesions incorporatedlarge voids of necrotic tissue that involved most of the hemicordipsilateral to the impact site and that typically extended to thecontralateral hemicord. White matter tracts of the contralateralhemicord were typically disrupted. By contrast, lesions inglibenclamide-treated animals were smaller, typically did not cross themidline, and contralateral as well as portions of ipsilateral whitematter tracts were spared. Lesion volumes at 7 d were ˜3-fold smaller inglibenclamide-treated rats compared to controls (FIG. 4D). Notably, thelesion volumes we observed with glibenclamide following a “severe”impact (10 gm×25 mm) were comparable to those observed by otherinvestigators in untreated rats using the same cervical contusion modelfollowing a “mild” impact (10 gm×6.25 mm) (Gensel et al., 2006).

Example 5 Glibenclamide Block of SUR1—Neurobehavioral Function

Vehicle-treated rats were generally not mobile (Soblosky et al., 2001),whereas glibenclamide-treated rats were typically ambulatory and oftenexhibited proficient exploratory behavior. When suspended by their tail,vehicle-treated rats hung passively with little or no flexion of thetrunk, whereas glibenclamide-treated rats could typically flex theirtrunk, bringing the snout to the level of the thorax or hindquarters.

The same animals used to assess lesion size on an inclined plane weretested, which is a standard test that requires more-and-more dexterousfunction of the limbs and paws as the angle of the plane is increased(Rivlin and Tator, 1977). At 1, 3 and 7 d post-SCI,glibenclamide-treatment was associated with significantly betterperformance than vehicle-treatment (FIG. 4E).

Ipsilateral paw placement was quantified, which is characteristicallylost following cervical hemicord transection (Nikulina et al., 2004). Inthe same animals tested 1 d post-SCI, glibenclamide-treatment wasassociated with significantly better performance than vehicle-treatment(FIG. 4E).

The BBB scale (Basso et al., 1995) is commonly used to evaluateneurobehavioral function in rodents post-SCI. However, it was designedfor thoracic-level lesions, not cervical-level lesions, and the highestlevel of performance that it records is less than what ourglibenclamide-treated rats could achieve. The vertical exploratorybehavior (“rearing”) was quantified, a complex exercise that requiresbalance, truncal stability, bilateral hindlimb dexterity and strength,and at least unilateral forelimb dexterity and strength, which togetherare excellent markers of cervical spinal cord function. Testing the samerats as above at 1, 3 and 7 d post-SCI showed thatglibenclamide-treatment was associated with significantly betterperformance than vehicle-treatment (FIG. 4E). In additional groups ofrats tested only at 1 d post-SCI, similar differences were observed (3±1vs. 42±7 sec; P=0.001; 14-15 rats/group).

Example 6 Repaglinide Block of SUR1

Repaglinide is a member of a distinct class of insulin secretagoguesthat are structurally unrelated to sulphonylureas and whose binding sitemay differ from that of sulfonylureas (Hansen et al., 2002). Likeglibenclamide, repaglinide produces high-affinity block of both nativeand recombinant β-cell K_(ATP) channels (IC₅₀=0.9-7 nM), and showshigher potency in inhibiting pancreatic SUR1-regulated KATP channelsthan cardiovascular SUR2-regulated channels (Stephan et al., 2006).

The effect of repaglinide on PHN was examined, using the same treatmentregimen as used for glibenclamide. As with glibenclamide, repaglinidetreatment reduced blood in cord homogenates from 1.8±0.2 to 1.2±0.1 alat 1 d post-SCI (P<0.01; 5-8 rats/group), and was associated withsignificantly better performance on the inclined plane (head up: 40±4vs. 62±2 degrees; P=0.01; head down: 29±4 vs. 47±3 degrees; P=0.03;n=3-8/group) and in vertical exploration (3±2 vs. 27±6 sec. P=0.005; 5-6rats/group) than vehicle-treated controls.

Example 7 Gene Suppression of SUR1

Gene suppression was used to confirm involvement of SUR1 in PHN,choosing an antisense-oligodeoxynucleotide strategy shown to beeffective in vitro (Yokoshiki et al., 1999).

To validate the antisense strategy, it was first implemented in themodel that was originally used for the discovery of the NC_(Ca-ATP)channel, wherein a gelatin sponge is implanted into the parietal lobe tostimulate formation of a gliotic capsule (Chen and Simard, 2001). Here,animals were also fitted with mini-osmotic pumps that deliveredoligodeoxynucleotides (ODN), either antisense (AS) or scrambled (Scr),continuously for 7 d into the injury site. Gliotic capsules from ratstreated with AS-ODN showed a significant reduction in SUR1 protein,compared to Scr-ODN (FIG. 5A). Patch clamp of astrocytes from glioticcapsule of rats treated with Scr-ODN showed that they rapidlydepolarized when cellular ATP was depleted by exposure to Na azide (FIG.5B), an effect that was previously shown was due to opening of NCCa-ATPchannels (Chen et al., 2003). By contrast, astrocytes from rats treatedwith AS-ODN depolarized only slightly or not at all (FIG. 5B),demonstrating that SUR1 is required for expression of functionalNC_(Ca-ATP) channels, just as with K_(ATP) channels (Sharma et al.,1999).

For experiments with SCI, AS-ODN and Scr-ODN were used that werephosphorothioated at 4 distal bonds to protect against endogenousnucleases (Galderisi et al., 1999), with ODN's administered i.v.starting immediately after injury. At 6 h post-SCI, cords from ratstreated with AS-ODN showed significantly less immunolabeling for SUR1than controls (FIG. 5C). With Scr-ODN, the necrotic void beneath theimpact site was surrounded by an SUR1-positive shell of tissue, similarto observations in untreated animals (FIG. 1A). With AS-ODN, however,only the small volume of tissue immediately beneath the impact site waslabeled for SUR1, and no necrotic void was evident (FIG. 5C). AS-ODN didnot affect normal expression of SUR1 in dorsal horn cells (FIG. 5C). At1 d post-SCI, treatment with AS-ODN reduced blood in cord homogenates,and was associated with significantly better performance on the inclinedplane and in vertical exploration compared to Scr-ODN (FIG. 5D).

Example 8 Significance of Certain Embodiments of the Invention

The present invention includes the novel finding that SUR1 is stronglyup-regulated following SCI, and that block of SUR1 is associated withsignificant improvements in all of the characteristic manifestations ofPHN, including hemorrhage, tissue necrosis, lesion evolution andneurological dysfunction. Although one embodiment focused on SUR1 andNC_(Ca-ATP) channels in capillary endothelium, the data also showedearly (<6 h) up-regulation of SUR1 in large neuron-like cells in thecore near the impact site, and in other studies, late (12-24 h)up-regulation of SUR1 in reactive astrocytes was observed. Theseresponses to SCI may be compared to findings previously reported forischemic stroke, wherein there is early up-regulation of SUR1 in neuronsand capillaries in the core, and later up-regulation of SUR1 incapillaries and astrocytes in penumbral tissues (Simard et al., 2006).

PHN has been linked to tissue ischemia (Nelson et al., 1977; Tator,1995), but has not previously been characterized at a molecular level.PHN is probably a variant of “hemorrhagic conversion”, a mechanism ofsecondary injury in the CNS, wherein capillaries or post-capillaryvenules undergo delayed catastrophic failure that allows extravasationof blood to form petechial hemorrhages, which in turn coalesce into aunified region of “hemorrhagic necrosis” or “hemorrhagic infarction”(Simard et al., 2007). Hemorrhagic conversion is common in traumaticbrain injury (Oertel et al., 2002) and following post-ischemicreperfusion (Wang et al., 2004), with hypoxia and active perfusion beingimportant antecedents (Simard et al., 2007). The molecular pathologyinvolved in hemorrhagic conversion has not been fully elucidated, butwork in ischemic stroke has implicated enzymatic destruction ofcapillaries by matrix-metalloproteinases (MMP) (Wang et al., 2004;Gidday et al., 2005). MMPs have been implicated in SCI (Noble et al.,2002; Pannu et al., 2007), but not in PHN.

The work reported here indicates that endothelial SUR1-regulatedNC_(Ca-ATP) channels are involved in PHN. The data show that PHN wasassociated with up-regulation of SUR1 in capillaries and post-capillaryvenules, structures long held to be responsible for PHN (Griffiths etal., 1978; Kapadia, 1984). Moreover, the data show that block of SUR1 by3 molecularly distinct agents, glibenclamide, repaglinide and AS-ODN,significantly reduced PHN. The remarkably similar outcomes obtained withhighly selective agents that act via distinct molecular mechanismsunderscore the important role of SUR1. These data also provide evidencethat de novo expression of SUR1 is necessary and sufficient fordevelopment of PHN. Use of a knock-down strategy employing AS-ODNappears to have been more informative than a gene knock-out strategy,since the latter would not have distinguished between constitutive andde novo expression of SUR1.

SUR1 forms the regulatory subunit of both NC_(Ca-ATP) and some K_(ATP)channels (Chen et al., 2003; Simard et al., 2006). Here, it is shownthat up-regulation of SUR1 in endothelial cells was associated withexpression of functional NC_(Ca-ATP) channels, which was previouslyimplicated in edema formation and cell death in CNS ischemia/hypoxia(Simard et al., 2006; Simard et al., 2007). Our patch clamp recordingsconfirmed the presence of non-selective cation channel that wasactivated by diazoxide and ATP-depletion, blocked by glibenclamide andcytoplasmic ATP, conducted Cs⁺, and had a single channel conductance of˜35 pS, all of which are characteristic of the NC_(Ca-ATP) channel (Chenand Simard, 2001; Chen et al., 2003). It was previously shown that thischannel conducts only monovalent, not divalent cations (Chen and Simard,2001). The studies reported here showing up-regulation of functionalNC_(Ca-ATP) channels were performed using endothelial cells from CNS aswell as non-CNS sources from two species, suggesting a certain degree ofgenerality of the phenomenon. In the patch clamp studies, endothelialcells from spinal cord were not explicitly studied, which couldpotentially differ from those in brain. However, it seems unlikely thatthe up-regulation of SUR1 in spinal cord capillaries that was observedwas associated with a different channel, such as K_(ATP). Sulfonylureablock of K_(ATP) would not be expected to be neuroprotective (Sun etal., 2007), whereas block of NC_(Ca-ATP) is highly neuroprotective inboth rodents and humans (Simard et al., 2006; Kunte et al., 2007).

Of the numerous treatments assessed in SCI, very few have been shown toactually decrease the hemorrhage and tissue loss associated with PHN.Methylprednisolone, the only approved therapy for SCI, improves edema,but does not alter the development of PHN (Merola et al., 2002). Anumber of compounds have shown beneficial effects related to tissuesparing, including the NMDA antagonist, MK801 (Faden et al., 1988), theAMPA antagonist, GYKI 52466 (Colak et al., 2003), Na⁺ channel blockers(Schwartz and Fehlings, 2001), and minocycline (Teng et al., 2004).Overall however, no treatment has been reported that reduces PHN andlesion volume, and that improves neurobehavioral function to the extentobserved here with glibenclamide, repaglinide and AS-ODN.

There are 2 mechanisms by which glibenclamide can antagonizingSUR1-regulated NC_(Ca-ATP) channels: (i) by block of the channel once itis expressed and subsequently opened by ATP depletion (Chen et al.,2003); (ii) by interfering with trafficking of SUR1 to the cellmembrane, a process that is required for expression of functionalchannels (Partridge et al., 2001). Both block of open channels (Simardet al., 2006) and SUR1 binding (Nelson et al.,) needed to inhibittrafficking are increased an order of magnitude or more at the low pH ofischemic tissues. Either block of open channels or interference withtrafficking or both, coupled with the augmented efficacy imparted by lowpH, likely account for the high efficacy of glibenclamide foundpreviously with stroke (Simard et al., 2006) and here with SCI.

Half of patients with SCI initially present with an incomplete lesion(Bracken et al., 1990), making it important to identify therapeuticstrategies to inhibit secondary injury mechanisms. Glibenclamide hasbeen used safely in humans for several decades for treatment of type 2diabetes, with no untoward side-effects except hypoglycemia, and itscontinued use immediately post-stroke improves outcome in patients withtype 2 diabetes (Kunte et al., 2007). The safety of glibenclamide, plusits unique mechanism of action in targeting the capillary failure thatleads to PHN, indicate that this drug may be especially attractive fortranslational use in human SCI.

Example 9 Exemplary Materials and Methods

SCI injury model. This study was performed in accordance with theguidelines of the Institutional Animal Care and Use Committee. Adultfemale Long-Evans rats (275-350 gm) were anesthetized (Ketamine, 60mg/kg plus Xylazine, 7.5 mg/kg, i.p.). The dura at C4-5 was exposed viaa left hemilaminectomy. A hemi-cervical spinal cord contusion wascreated using a blunt force impactor (1.3-mm impactor head driven by a10 gm weight dropped vertically 25 mm) (Soblosky et al., 2001; Gensel etal., 2006). After SCI, animals were given 10 ml of glucose-free normalsaline s.q. Rectal temperature was maintained at ˜37° C. using aservo-controlled warming blanket. Blood gases and serum glucose 10-15min post-SCI were: pO₂, 95±6 mm Hg; pCO₂, 46±3 mm Hg; pH, 7.33±0.01glucose 258±17 mg/dl in controls and pO₂, 96±7 mm Hg; pCO₂, 45±2 mm Hg;pH, 7.37±0.01; glucose 242±14 mg/dl in glibenclamide-treated animals.

Drug delivery. Within 2-3 min post-SCI, mini-osmotic pumps (Alzet 2002,0.5 μl/h; Durect Corporation) were implanted that delivered eithervehicle (saline plus DMSO), glibenclamide (Sigma) in vehicle, orrepaglinide (Sigma) in vehicle subcutaneously. During the course of theproject, slightly different formulations of drug were used, with thebest results obtained using stock solutions made by placing 50 mg (or 25mg) of drug into 10 ml DMSO, and infusion solutions made by placing 400μl (or 800 μl) stock into 4.6 ml (or 4.2 ml) unbuffered saline (0.9%NaCl) and adjusting the pH to ˜8.5 using 0.1 N NaOH. Infusion solutionsof glibenclamide and repaglinide were delivered at 0.5 μl/h, yieldinginfusion doses of 200 ng/h.

For in vivo gene suppression of SUR1, we used oligodeoxynucleotides thatwere phosphorothioated at 4 distal bonds to protect against endogenousnucleases (35). Within a few min of SCI, mini-osmotic pumps (Alzet 2002,0.5 μl/h; Durect Corporation) with jugular vein catheters were implantedthat delivered either scrambled sequence ODN (Scr-ODN)(5′-TGCCTGAGGCGTGGCTGT-3′; SEQ ID NO:1) or antisense ODN (AS-ODN)(5′-GGCCGAGTGGTTCTCGGT-3′; SEQ ID NO:2) (Yokoshiki et al., 1999) in PBSat a rate of 1 mg/rat/24 h.

Tissue blood. Rats were sacrificed at various times after SCI (n=5-11rats/group), were perfused with heparinized saline to removeintravascular blood, and 5-mm segments of cord encompassing the lesionwere homogenized and processed as described (Choudhri et al., 1997).

Lesion size. At 7 d post-SCI, cords were paraffin sectioned and stainedwith H&E. Lesion volumes were calculated from lesion areas measured onserial sections every 250 μm.

Neurobehavioral assessment. All measurements were performed by blindedevaluators. Performance on the inclined plane was evaluated as described(Rivlin and Tator, 1977). To assess paw placement and verticalexploration (rearing) (Nikulina et al., 2004) animals were videotapedwhile in a translucent cylinder (19×20 cm). Rearing was quantified asthe number of seconds spent with both front paws elevated aboveshoulder-height during a 3-min period of observation.

Bleeding times were measured using tail tip bleeding as described(Lorrain et al., 2003).

Zymography of recombinant MMP-2 and MMP-9 (Sigma) was performed asdescribed (Sumii and Lo, 2002).

Cell culture. Endothelial cell cultures from human brain microvessels,human aorta (ScienCell Research Laboratories), and murine brainmicrovessels (bEnd.3: ATCC), were grown at low density using media andsupplements recommended by suppliers.

SUR1 knock-down in astrocytes was performed in triplicates by implantingrats with gelatin sponges in the parietal lobe to induce formation of agliotic capsule containing reactive astrocytes that express theSUR1-regulated NC_(Ca-ATP) channel (Chen and Simard, 2001; Chen et al.,2003). At the same time, they were implanted with mini-osmotic pumps(Alzet, model 2002; 14-day pump) placed in the dorsal thoracolumbarregion that contained ODN (711 μg/ml delivered @ 0.5 μl/h, yielding 1500picomoles/day), with the delivery catheter placed directly into the siteof the gelatin sponge implant in the brain. Animals were infused withSrc-ODN or AS-ODN, as above but not phosphorothioated. After 10-14 days,the gelatin sponge plus encapsulating gliotic tissues were harvested andprocessed either for Western immunoblots or to obtain fresh reactiveastrocytes for patch clamp electrophysiology.

Patch clamp electrophysiology for the NC_(Ca-ATP) channel in this labhas been described (Chen and Simard, 2001; Chen et al., 2003).

Immunohistochemistry. Cryosections were immunolabeled (Chen et al.,2003; Simard et al., 2006) using primary antibodies directed againstSUR1 (Santa Cruz, C-16; 1:200; 1 h at RT, 48 h at 4° C.), SUR2 (SantaCruz, H-80; 1:200; 1 h at RT, 48 h at 4° C.), GFAP (Sigma, C-9205;1:500), and vimentin (Sigma, monoclonal CY3 conjugated; 1:100).Quantitative immunofluorescence was performed as described (Gerzanich etal., 2003).

Immunoblots were prepared using antibodies directed against SUR1. Thespecificity of the antibody (Chen and Simard, 2001; Chen et al., 2003;Simard et al., 2006) is demonstrated by the knock-down experiments ofFIG. 5.

In situ hybridization. Fresh-frozen cord sections were fixed in 5%formaldehyde for 5 min. Digoxigenin-labeled probes (sense:′5-GCCCGGGCACCCTGCTGGCTCTGTGTGTCCTTCCGCGCCTGGGCATCG-3′; SEQ ID NO:3)were designed and supplied by GeneDetect and hybridization was performedaccording to the manufacturer's protocol (see website for GeneDetect).

Example 10 Spinal Cord Injury, Progressive Hemorrhagic Necrosis and theNC(Ca-ATP) Channel

Anti-SUR1 antibody. Because of the emerging importance of theSUR1-regulated NC_(Ca-ATP) channel in SCI and other disorders (Simard etal., 2007), an antibody against SUR1 was developed. A part of the ratSUR1 cDNA (Protein Id, NP_037171; amino acid 598-965) was subcloned intopQE31 (Qiagen, Chatsworth, Calif.) to overexpress the protein in ahexa-histidine-tagged form in bacterial cells. The fusion protein waspurified using a Ni+-agarose column and was used to raise antibodies inrabbits by a commercial service (Covance, Denver, Pa.). To validate theantibody, flag-tagged SUR1 was expressed in COS7 cells. Total lysatesfrom COS7 cells transfected with a control empty vector (FIG. 6A lane 1,FIG. 6B lane 1) or with an expression vector encoding FLAG-tagged SUR1(FIG. 6A lanes 2 and 3, FIG. 6B lanes 2 and 3) were examined byimmunoblot using FLAG monoclonal M2 antibody (FIG. 6A) and the anti-SUR1polyclonal antibody generated in this lab (FIG. 6B). Both antibodiesdetected the same band at ˜160 kDa, as well as higher molecular weightproducts believed to be due to poly-ubiquitination of SUR1, as reportedpreviously (Yan et al., 2005) Note that neither antibody detected anyspecific band from lysates from cells transfected with a control vector(FIG. 6A lane 1, FIG. 6B lane 1), consistent with a high specificity ofthe antibody.

Exemplary data on human SCI. Because of the emerging importance of theSUR1-regulated NC_(Ca-ATP) channel in SCI and other disorders (Simard etal., 2007) the upregulation of the channel in human SCI wasinvestigated. To date, SUR1 expression has been studied in 3 human casesusing the antibody referred to above. The exemplary case illustratedhere is that of a 59 yo male who sustained a C3 level injury and expired3 days later. Low power views of H&E sections at the level of injuryshowed gross tissue disruption, which was not present in “uninvolved”cord (FIG. 7A vs. FIG. 7B). Immunolabeling of adjacent sectionsdemonstrated diffuse upregulation of SUR1 throughout the area ofinvolvement (FIG. 7C vs. FIG. 7D). High power views of H&E sectionsconfirmed the presence of extravasated blood and fractured microvesselswithin the core of the lesion, but not in “uninvolved” cord (FIG. 7E vs.FIG. 7F), and confirmed the presence of dying neurons in the core butnot in “uninvolved” cord (FIG. 7G vs. FIG. 7H). Sections from the coreshowed prominent expression of SUR1 in microvessels (FIG. 8A, arrows),in ballooned neurons (FIG. 8B), in microvascular endothelium (FIG. 8C,arrow) and in endothelium of arterioles (FIG. 8C, *□ and FIG. 8D,arrows). Each of these findings in humans duplicates exactly recentfindings in rats (Simard et al., 2007). Notably, SUR1 is not normallyexpressed in CNS microvessels (Sullivan and Harik, 1993), making thesefindings in human microvessels post-SCI remarkably similar to thefindings in rodents. In specific embodiments, double labeling of thesetissues is employed to verify cellular identity and in situhybridization to confirm SUR1 upregulation. Nevertheless, these excitingfindings indicate that progressive secondary hemorrhage in humans may beameliorated, as in rodents, by block of SUR1 with glibenclamide.

Exemplary data on SCI in SUR1-KO mice. A colony of SUR1-KO mice ismaintained to perform studies to demonstrate the beneficial effect ofSUR1-KO in SCI. An active colony of >20 SUR1-KO mice that aresuccessfully breeding now exists. Additional SCI experiments have beenperformed (unilateral T9 lesion). The behavioral response was evaluatedat 24 hr in 14 WT and in 18 SUR1-KO mice using BMS, confirming thatSUR1-KO is highly protective against progressive hemorrhagic necrosis(FIG. 9). In addition, longer term outcome in investigated, for exampleto assess durability of the protective effect. Data at 7 days continueto show highly significant differences between WT and SUR1-KO.

In certain embodiments of the invention, transfection of plasmids intoendothelial cells, both bEnd.3 cells and primary cultured CNSmicrovascular endothelial cells, is employed. To improve transfectionefficiencies, the Nucleofector 96-well shuttle system is utilized. Twoexperiments were performed with transfection of plasmids that encodeGFP: 1) with primary cultured CNS microvascular cells, there was asurvival rate of 30% at 24 hrs, with 90% of surviving cells showingfluorescent signal; 2) with bEnd.3 cells, there was a survival rate of60% at 24 hrs, with >70% of surviving cells showing fluorescent signal.The transfection parameters to improve cell survival rates with thismethod were optimized.

Example 11 SUR1 Upregulation Predisposes Premature Infants toIntraventricular Hemorrhage

Brain tissues were obtained at autopsy from 6 premature infants with IVHand 3 controls without IVH. For routine histopathology, sections ofgerminal matrix in affected areas were dehydrated in graded ethanol andxylene solutions, embedded in paraffin, and sectioned at 6 microns. Forimmunofluorescence for SUR1, fixed and unprocessed tissues weresuspended in sucrose and snap frozen. Six micron cryostat sections wereobtained. Immunofluorescence for SUR1 was performed as previouslydescribed (Nature Medicine 2006; 12:433-40).

Significant increased expression of SUR1 was observed in vascularendothelium and germinal matrix tissue in one of the three non-IVHcases; the clinical course of this case was complicated by hypoxianecessitating intubation. A second non-IVH case showed an intermediatelevel of fluorescence in germinal matrix only; this infant expired ofextreme prematurity shortly after delivery. The third non-IVH alsoexpiring of extreme prematurity following delivery, showed noreactivity. The 6 IVH cases showed patchy increased fluorescenceconsistent with up-regulation followed by early ischemic necrosis.

These results indicate that SUR1 is increased in premature infantbrains, and particularly in germinal matrix regions of infants whosuffer hypoxia and IVH. This suggests that maladaptive opening of theNC_(Ca-ATP) channels may result in endothelial injury and hemorrhage.Since SUR1 is blocked at least by glibenclamide, these data provide auseful therapeutic and/or preventative intervention in prematureinfants, including stressed premature infants prior to IVH.

Example 12 In Utero Ischemia Leads to the Upregulation of SulfonylureaReceptor 1 in the Periventricular Zone in Rats

Periventricular leukomalacia (PVL) is a form of cerebral palsy thatinvolves deep white matter injury and that usually occurs during fetaldevelopment. In specific embodiments of the invention, hypoxic/ischemicinsults during pregnancy induces the expression of sulfonylurea receptor1 (SUR1)-regulated NC(Ca-ATP) channels, which were recently implicatedin programmed oncotic cell death in the central nervous system (CNS),and have been found to play an important role in cerebral ischemia andspinal cord injury. In this study, expression of the regulatory subunitof the channel, SUR1, was evaluated in a rodent model of prenatalischemia/hypoxia. Transient (1 hr) unilateral uterineischemia/reperfusion was induced in pregnant rats at embryonic day 17 byclamping the right uterine artery. Embryos in the left uterine horn,where blood flow was not interrupted, served as controls.

Embryos were delivered by cesarean section 24 hr after uterineischemia/reperfusion. SUR1 was prominently upregulated in the brains ofembryos that were subjected to ischemia/reperfusion, but not incontrols.

Especially prominent upregulation of SUR1 was found in neural progenitorcells in the subventricular zone, which corresponds to the area ofvulnerability that is affected in PVL. Additionally, neurons in thecortex of ischemic embryos exhibited increased SUR1 compared to controlembryos. Thus, in certain aspects of the invention, SUR1 is upregulatedfollowing intrauterine transient ischemia. In specific embodiments, itis determined whether the pore-forming subunit of the SUR1-regulatedNC(Ca-ATP) is also upregulated, and whether this novel pathologicalmechanism accounts for PVL following intrauterine ischemia/hypoxia.

Example 13 In Utero Ischemia Upregulates SUR1—Links to PeriventricularLeukomalacia and Germinal Matrix Hemorrhage

Premature infants often suffer from cerebral palsy (CP), which leads todevastating lifelong disability. At present, there is no good preventionfor CP. CP is believed to arise from periods of reduced blood flow tothe brain in utero, which predisposes premature infants to white matterinjury (periventricular leukomalacia) and bleeding in the brain(germinal matrix hemorrhage) during the early post-natal period. Theexperiments reported here were intended to model this condition in rats.Using pregnant rats, the uterine artery was temporarily clamped on oneside to mimic placental insufficiency. The next day, the pups weredelivered “prematurely” by C-section. Shortly after birth, saline wasinjected into the abdomen of the pups to raise central venous pressure,to mimic complications associated with mechanical ventilation oftenrequired in premature infants with “stiff” lungs. The pups were latereuthanized, within 1 hr of birth. The pups from the opposite side, wherethe uterine artery was not clamped, were used as controls. The brains ofthe pups were studied to detect the regulatory subunit of the SUR1regulated NC_(Ca-ATP) channel. SUR1 was found to be significantlyupregulated in periventricular progenitor cells and in veins, consistentwith the embodiment that SUR1-regulated NC_(Ca-ATP) channels may becausally linked to the brain damage in humans characterized asperiventricular leukomalacia and germinal matrix hemorrhage.

Introduction

The neuropathology underlying cerebral palsy includes white matterinjury, known as periventricular leukomalacia (PVL) and germinal matrix(GM) hemorrhage (GMH) (Vergani et al., 2004; Folkerth, 2005). Each hasdistinctive features, but both share important risk factors, includingprematurity and hypoxia/ischemia, which may occur prenatally or may bedue to post-natal ventilatory difficulties that are complicated bymild-to-moderate hypotension (Veragni et al., 2004; Kadri et al., 2006;Lou, 1993).

GMH is a common complication of prematurity, occurring in 20-45% ofpremature infants (Kadri et al., 2006). GMH may range in severity fromsubependymal hemorrhage (grade 1) to intraventricular hemorrhage without(grade 2) or with (grade 3) ventricular dilatation, to parenchymalextension and periventricular venous infarction (grade 4). In survivors,neurological sequelae, particularly with higher grade GMH, includecerebral palsy, hydrocephalus requiring ventricular shunting, learningdisabilities, and seizures (Levy et al., 1997; Pikus et al., 1997).Numerous factors are believed to contribute to GMH, including innateweakness of GM veins, autoregulatory dysfunction, hypoxic/ischemictissue damage, damage due to post-ischemic reperfusion and increasedvenous pressure (Lou, 1993; Nakamura et al., 1990; Wei et al., 2000;Anstrom et al., 2004; Berger et al., 2002; Ghazi-Birry et al., 1997).The incidence of GMH increases with the degree of prematurity (Kadri etal., 2006), suggesting that advances in perinatal care that yieldconcomitant increases in the number of extremely premature infants willcontinue to be hampered by complications of GMH. At present, noeffective prevention is available.

Hypoxia/ischemia in human CNS, both in utero (Xia et al., 1993) and inadults (Xia et al., 1993; Simard et al., 2007) results in upregulationof sulfonylurea receptor 1 (SUR1). Under pathological conditions, SUR1upregulation is associated with formation of SUR1-regulated NC_(Ca-ATP)channels, not K_(ATP) channels (Simard et al., 2006; Simar et al.,2007a; Simard et al., 2007b). Expression of SUR1-regulated NC_(Ca-ATP)channels in capillary endothelium has been causally implicated inprogressive secondary hemorrhage in CNS, with block of these channels byinfusion of low-dose (non-hypoglycemogenic) glibenclamide (glyburide)completely preventing secondary hemorrhage (Simard et al., 2007b). Inspecific embodiments, this channel is induced in periventriculartissues, including the GM, by hypoxia/ischemia, and thereby predisposeto PVL and GMH. To assess this, expression of the regulatory (SUR1)subunit of the channel in brain tissues was studied from a rat model ofintrauterine ischemia.

Methods

Pregnant female Wistar rats were shipped to arrive on gestational day(GD) (Simard et al., 2006; Simard et al., 2007b; Simard et al., 2007b).They were acclimatized, then on GD 17, they underwent surgery fortemporary clamping of the right uterine artery. An animal wasanesthetized to a surgical level with 3% isoflurane in the mixtureN₂O/O₂, 70° %/30%, after which anesthesia is maintained with 1.5%isoflurane during surgery. Core temperature is maintained at 37° C.Transient unilateral uterine ischemia was induced as described (Nakai etal., 201; Tanaka et al., 1994). Two sterile microvascular clips wereused to occlude the uterine vessels near the lower and upper ends of theright uterine horn. The clips were removed after 60 min of ischemia. Foreach experiment the fetuses in the right uterine horn served as theischemia group and those in the left horn as the non-ischemia group.

24 hr after induction of uterine ischemia, the rats werere-anesthetized. The fetuses are delivered by cesarean section, afterwhich the dam was euthanized. All the pups delivered from the left cornu(non-ischemic side) and half the pups delivered from the right cornu(ischemic side) underwent no further intervention. The other half of thepups from the right cornu (ischemic side) underwent a singleintraperitoneal injection of sterile, USP grade normal saline (100 μl).One hr after birth, all pups were euthanized for tissue analysis.Results

Immunolabeling of brains from control pups showed no appreciable SUR1.However, pups subjected to transient ischemia/hypoxia showed significantupregulation of SUR1, especially in the progenitor cells that weredensely packed in periventricular regions (FIG. 10A). In pups exposed totransient ischemia/hypoxia plus an increase in central venous pressure,SUR1 was also found to be prominently upregulated in veins (FIGS.10B-10D).

Conclusions

SUR1 is upregulated in periventricular progenitor cells in a rodentmodel of in utero ischemia/hypoxia and, when central venous pressure isincreased, in veins as well. This pattern of SUR1 upregulationcorresponds to the pattern observed in premature infants at risk for orwho sustain germinal matrix hemorrhages. The known functions of theSUR1-regulated NC_(Ca-ATP) indicate that SUR1 upregulation following inutero ischemic/hypoxic insults is causally linked to pathologicaldisorders such as periventricular leukomalacia and germinal matrixhemorrhage, for example.

Example 14 Sulfonylurea Receptor 1 in the Germinal Matrix of PrematureInfants

The present example concerns germinal matrix (GM) hemorrhage (GMH),which is a major cause of mortality and of life-long morbidity fromcerebral palsy (CP). GMH is typically preceded by hypoxic/ischemicevents and is believed to arise from rupture of weakened veins in theGM. In the CNS, hypoxia/ischemia upregulate sulfonylurea receptor 1(SUR1)-regulated NC_(Ca-ATP) channels in microvascular endothelium, withchannel activation by depletion of ATP being responsible for progressivesecondary hemorrhage. In specific embodiments of the invention, thischannel is upregulated in the GM of preterm infants at risk for GMH.Here, the expression of the regulatory subunit of the channel, SUR1, andits transcriptional antecedent, hypoxia inducible factor 1 (HIF1), wereexamined in postmortem tissues of premature infants who either were atrisk for or who sustained GMH. Regionally specific upregulation of HIF1and of SUR1 protein and mRNA in GM tissues was identified, compared toremote cortical tissues. Upregulation was prominent in most progenitorcells, whereas in veins, SUR1 was found predominantly in infants who hadsustained GMH compared to those without hemorrhage. The data indicatethat the SUR1-regulated NC_(Ca-ATP) channel is associated with GMH, incertain embodiments, and that pharmacological block of these channelsreduces the incidence of this devastating complication of prematurity.

The neuropathology underlying cerebral palsy includes white matterinjury, such as periventricular leukomalacia (PVL) and germinal matrix(GM) hemorrhage (GMH) (Vergani et al., 2004; Folkerth, 2005). Each hasdistinctive features, but both share important risk factors, includingprematurity and hypoxia/ischemia, which may occur prenatally or may bedue to post-natal ventilatory difficulties that are complicated bymild-to-moderate hypotension (Vergani et al., 2004; Kadri et al., 2006;Lou, 1993).

GMH is a common complication of prematurity, occurring in 15-45% ofpremature infants (Kadri et al., 2006). GMH may range in severity fromsubependymal hemorrhage (grade 1) to intraventricular hemorrhage without(grade 2) or with (grade 3) ventricular dilatation, to periventricularvenous infarction (grade 4). In survivors, neurological sequelae,particularly with higher grade GMH, include cerebral palsy,hydrocephalus requiring ventricular shunting, learning disabilities, andseizures (Levy et al., 1997; Pikus et al., 1997). Numerous factors arebelieved to contribute to GMH, including innate weakness of GM veins,autoregulatory dysfunction, hypoxic/ischemic tissue damage, damage dueto post-ischemic reperfusion and increased venous pressure (Lou, 1993;Nakamura et al., 1990; Wei et al., 2000; Anstrom et al., 2004; Berger etal., 2002; Ghazi-Birry et al., 1997). The incidence of GMH increaseswith the degree of prematurity (Kadri et al., 2006), suggesting thatadvances in perinatal care that yield concomitant increases in thenumber of extremely premature infants will continue to be hampered bycomplications of GMH. At present, no effective prevention is available.

Hypoxia/ischemia in rodent and human CNS, both in utero (Xia et al.,1993) and in adults (Simard et al., 2006; Simard et al., 2008), resultsin upregulation of sulfonylurea receptor 1 (SUR1). Under pathologicalconditions, SUR1 upregulation is associated with formation ofSUR1-regulated NC_(Ca-ATP) channels, not K_(ATP) channels (Simard etal., 2006; Simard et al., 2008; Simard et al., 2007). Expression ofSUR1-regulated NC_(Ca-ATP) channels in capillary endothelium has beencausally implicated in progressive secondary hemorrhage in CNS, withblock of these channels by infusion of low-dose (non-hypoglycemogenic)glibenclamide (glyburide) completely preventing secondary hemorrhage(Simard et al., 2007). Here, in certain embodiments, this channel isinduced in the GM by hypoxia/ischemia, and thereby predisposes one toGMH. As an initial attempt to assess this embodiment, expression of theregulatory subunit of the channel, SUR1, and its transcriptionalantecedent, hypoxia inducible factor 1 (HIF1) was studied (Bhatta, 2007)in postmortem tissues of premature infants who either were at risk foror who sustained GMH. The findings are consistent with the embodimentthat the SUR1-regulated NC_(Ca-ATP) channel is causally linked to GMH.

Methods

Specimens from premature infants without and with clinically diagnosedGMH were obtained from the Brain and Tissue Bank for DevelopmentalDisorders, University of Maryland, Baltimore, with the collectionprotocol, including informed consent, reviewed and approved by theInstitutional Review Board of the University of Maryland at Baltimore.The post-mortem interval was 3-24 hr. Cases were selected for studybased either on: (i) the documented presence of GMH/IVH at autopsy or(ii) documented absence of GMH (used as “best-available” controls).Independent histological validation of presence or absence of GMH wasmade in all cases (see Table 1). In all but one case, the cause ofprematurity was preterm rupture of membranes, with some cases alsodocumenting chorioamnionitis by pathological examination of theplacenta, and one case (without GMH) being induced for cardiac anomaly.The cause of death was extreme prematurity in all but two cases, withthe others being listed as amniotic fluid aspiration syndrome orelective termination.

TABLE 1 Summary of exemplary cases examined. Gestational HIF1 Age @Hemorrhage* protein SUR1 SUR1 birth Hemorrhage in in protein proteinCase # (weeks) clinically H&E section cells** in cells^(§) in veins^(¶)1 19 none none + +++ 0 2 19 none none ++ + 0/+ 3 22 none none ++ +++ + 422 none none +++ ++ + 5 22 none none +++ + +/++ 6 23 none microscopic++ + +/++ 7 24 none microscopic +++ +++ +/++ 8 24 grade 1 grade 1 ++++++ ++++ 9 22 grade 1 grade 1 ++++ ++++ ++++ 10 24 grade 2/3 none ++++++++ ++++ 11 30 grade 2/3 grade 1 +++++ +++ ++++ 12 23 grade 2/3 grade2/3 +++++ ++ +++ *clinical information was available only on“intraventricular hemorrhage” without differentiating further intograde, hence the designation, grade 2/3; some discrepancies in clinicalvs. histological evaluation of hemorrhage may be due to histologicalevaluation of the GM contralateral to the side of hemorrhage, whichavailable data were insufficient to resolve **scale for HIF1immunolabeling in progenitor cells within the GM: +, present in mostcells, similar in intensity to some distant neurons; ++, present in mostcells, somewhat more intense than in neurons; +++, present in mostcells, definitely more intense than in neurons; ++++, present in allcells, more intense than in neurons; +++++, present in all cells, manywith very intense labeling ^(§)scale for SUR1 immunolabeling, inprogenitor cells within the GM: +, present in few single cells; ++,present in a moderate number of scattered cells; +++, present in patchesor groups of cells; ++++ present in most cells scale for SUR1immunolabeling in veins within the GM: 0, none; +, in 1-2 veins; ++, ina few veins; +++, in many veins; ++++, in nearly all veins.

GM tissues and associated hemorrhages, when present, were dissected fromcoronal slices of formalin-fixed cerebral hemispheres. Cryosections andparaffin-embedded sections were prepared. Sections were stained withhematoxylin and eosin (H&E) or were immunolabeled using primaryantibodies directed against SUR1 (C-16; Santa Cruz Biotechnology Inc.;diluted 1:200; 1 hr at room temperature (RT), 48 hr at 4° C.), or HIF-1α(SC-10790; Santa Cruz; 1:100), or von Willebrand factor (F-3520; Sigma;1:200). CY-3 or FITC conjugated secondary antibodies (JacksonImmunoresearch, West Grove, Pa.) were used. Slides were cover slippedwith ProLong Gold antifade reagent containing4′,6-diamino-2-phenylindole (DAPI) for nuclear staining (P36935,Invitrogen, Carlsbad, Calif.). For in situ hybridization,digoxigenin-labeled probes (antisense,5′-TGCAGGGGTCAGGGTCAGGGcGCTGTCGGTCCACTTGGCCAGCCAGTA-3′; SEQ ID NO:4),designed to hybridize to nucleotides 3217-3264 located within codingsequence of human Abcc8 gene (NM_000352; GenBank@ Accession number forthe sequence, which is incorporated by reference herein in itsentirety), were supplied by GeneDetect (Brandenton, Fla.). Hybridizationwas performed according to the manufacturer's protocol, as previouslydescribed (Simard et al., 2006).

Results

The germinal matrix appeared as a dense collection of small cellslocated peri-ventricularly (FIG. 11A). In some cases, evidence of aparenchymal hemorrhage was found (FIG. 11A, arrow).

In situ hybridization for mRNA for Abcc8, which encodes SUR1, showedregionally specific upregulation in the GM (FIGS. 11B, 11E) that wasnoticeably more prominent than in surrounding tissues or in remotecortical tissues (FIG. 11D). Immunolabeling confirmed regionallyspecific upregulation of SUR1 protein in the GM (FIG. 11C), with SUR1protein located in neural progenitor cells in all GM specimens examined(FIG. 11G). SUR1 protein was also identified in veins from infants withGMH (FIGS. 11H, 11I), but was less likely to be found in veins frominfants without GMH (FIG. 11J). Negative controls, including omission ofprimary antibody and use of a blocking peptide, showed no immunolabelingfor SUR1 (not shown).

An important molecular antecedent of SUR1 is the transcription factor,HIF1 (Bhatta, 2007), which is upregulated by hypoxia (Wenger et al.,2005), a common condition associated with prematurity. Immunolabelingfor HIF1α showed that this ubiquitous marker of hypoxia was prominentlyupregulated, with characteristic nuclear localization, in all GMspecimens examined (FIGS. 11K-11M).

A semi-quantitative assessment was performed of HIF1α and SUR1expression in specimens from 12 premature infants, some of whom hadeither clinical or histological evidence of GMH (Table 1). All specimensshowed HIF1 α expression, with all but one showing more prominentexpression in progenitor cells than in remote neurons in the same tissuesections, supporting the embodiment that physiologically meaningfulhypoxia was present in the GM of all of these cases. The most prominentexpression of HIF1 α was found in specimens from infants with frank GMH.All specimens showed SUR1 expression in progenitor cells. In 3specimens, SUR1 was identified only in scattered cells, whereas in mostspecimens, SUR1 expression was evident in contiguous sheets of cells orin some cases, in nearly all cells. The clearest distinction in SUR1expression vis-à-vis GMH was in the veins of the GM. In specimenswithout GMH, the veins typically exhibited little to moderate SUR1expression, as in FIG. 11J, whereas in all specimens with frank GMH, allor nearly all veins exhibited strong SUR1 expression, as in FIGS. 11H,11I.

Significance of Certain Embodiments

Thus, expression of SUR1 is increased in neural progenitor cells and invascular endothelium of the GM of premature infants who either are atrisk for or who sustained GMH. Immunohistochemical analysis ofpost-mortem tissues can sometimes be complicated by non-specific bindingof antibodies, especially if necrosis is present. However, the specimensstudied showed intact cellular structures with H&E staining, as well asregionally-specific immunolabeling of cellular and vascular structuresfor SUR1 in the GM. Most importantly, in situ hybridization was used toconfirm that SUR1 was upregulated at the mRNA level. Together, the twoindependent techniques using molecularly distinct probes provideimportant corroborative evidence that SUR1 was upregulated in GM tissuesof premature infants. Additional work is performed to demonstrateconcomitant upregulation of the pore-forming subunit of the channel(Simard et al., 2008).

Pathophysiology. The pathophysiological antecedents of GMH have beenextensively discussed, but no fully encompassing theory has been putforth to explain it. Considerable attention has been focused on thestructural weakness of GM microvessels (Wei et al., 2000; Anstrom etal., 2004). However, it is evident that any innate weakness of thesevessels, by itself, would be insufficient to cause GMH, since the sameweakness exists during every gestation, and most gestations are notcomplicated by GMH. Thus, an event must transpire to weaken thesevessels further and increase the likelihood of their structural failure.In the premature brain, the GM is at the terminal end of its afferentarteriolar supply (“ventriculopetal” vascular pattern) (Nakamura et al.,1994) and therefore GM tissues and the vessels contained therein arehighly susceptible to global hypoxic/ischemic events. Apart from hypoxiadue to ventilatory abnormalities, one or more hypotensive episodes maycontribute to the overall hypoxic/ischemic burden that adversely affectsGM tissues. In addition, it is likely that yet another hemodynamicstress must be applied to structurally compromised vessels to cause anactual GMH. Because GMH most frequently arises from veins (Nakamura etal., 1990; Ghazi-Birry et al., 1997), it is thought that episodes ofincreased venous pressure, as can occur with mechanical ventilation orairway suctioning, may be important for triggering the actual structuralfailure of weakened vessels that results in GMH.

Despite the important role of hypoxia/ischemia in producing vascularchanges that predispose to GMH, there is little experimental evidence toelucidate the molecular mechanism involved, either in animal models orin humans. The present invention is the first report to show that thetranscription factor, HIF1, is upregulated in the GM of infants at risk.In many organs including the CNS, hypoxia results in activation of HIF1,which in turn stimulates the transcription of genes that are essentialfor adaptation to hypoxia/ischemia, including genes important forerythropoiesis, glycolysis and angiogenesis (Wenger et al., 2005). HIF1plays a critical role in expression of the angiogenic factor, vascularendothelial growth factor (VEGF), which is prominently upregulated inthe GM of infants at risk (Ballabh et al., 2007). Conversely, HIF1 alsocauses transcription of genes with seemingly maladaptive effects (Simardet al., 2007) and, in some settings, may promote ischemia-inducedneuronal death (Chang and Huang, 2006). HIF1 has not been extensivelystudied in the premature infant brain, and a role for HIF1 has notpreviously been suggested in the context of GMH. However, thelocalization of HIF1 with two of its important transcriptional targets,VEGF (Ballabh et al., 2007) and SUR1 (Bhatta, 2007), in the GM ofinfants at risk reaffirms the importance of this molecular response tohypoxia.

Events in the GM. Mild hypoxia activates quiescent neural progenitorcells, resulting in their activation and differentiation into neuronsand glia, whereas severe hypoxia induces apoptotic death in developingbrain neurons (Pourie et al., 2006). Thus, mild-to-moderate hypoxia,resulting from the position of the GM as the distant-most tissue fed bya ventriculopetal blood supply (Ballabh et al., 2007), may be involvednot only in stimulating neurogenesis from GM progenitor cells, but alsoin the normal involution of the GM (FIG. 12). HIF1, the ubiquitoussensor of hypoxia, may be a key molecular participant in both. Notably,the same hypoxic signal working via HIF1 also leads to transcriptionalupregulation of SUR1 (Bhatta, 2007) and of SUR1-regulated NC_(Ca-ATP)channels (Simard et al., 2007). In all of the 12 cases studied, most ofthe progenitor cells exhibited both HIF1 and SUR1, indicating that mildhypoxia may be a normal state in germinal matrix parenchyma, and thatthis tissue may be normally “primed” with SUR1. When the NC_(Ca-ATP)channel is expressed in response to an hypoxic stimulus, no adversefunctional consequence is expected, as long as intracellular ATP ismaintained at sufficient levels (>30 μM) to keep the channel fromopening (Simard et al., 2008).

Under conditions of extreme duress, a normal hypoxic signal may bemagnified by one or more ischemic events, leading to more profoundhypoxia. Under such conditions, HIF1 activation and SUR1 expressionwould become more likely, especially in veins (FIG. 12). Normally, cellsof the vascular tree are less likely than parenchymal cells toexperience hypoxia, but under conditions of extreme duress, when maximumextraction of O₂ has already occurred from hypoxic blood, venular cellswill experience the strongest hypoxic challenge. In the cases westudied, veins generally were less likely to exhibit SUR1 thanparenchymal cells, but in cases with GMH, SUR1 expression was reliablyfound in most veins—the very structures that are believed to be thesource of hemorrhage (Nakamura et al., 1990; Ghazi-Birry et al., 1997).When ATP is depleted to critical levels, SUR1-regulated NC_(Ca-ATP)channels open, leading to oncotic cell death (Simard et al., 2006) notonly of progenitor cells but of vascular endothelial cells, therebyfurther weakening thin walled, structurally compromised veins. In thissetting, increased venous pressure would almost certainly causeextravasation of blood from damaged veins. Petechial hemorrhages mayenlarge to microhemorrhages or grade 1 GMH, or worse, depending on theseverity and extent of GM tissues involved. In specific embodiments,this sequence (FIG. 12), which employs critical involvement of HIF1 andSUR1, accounts for numerous observations and encompasses numeroushypotheses that have been put forth to explain GMH.

Preventing GMH. Available strategies for preventing GMH are limited.Currently, the most effective measures are those that target therespiratory system (Cools and Offringa, 2005; Wright et al., 1995).Vitamin E, phenobarbital, morphine, ibuprofen, indomethacin, agents thattarget coagulation, and magnesium/aminophylline have been tried, but areeither ineffective or their use remains controversial. In an animalmodel of GMH, prenatal treatment with angiogenic inhibitors reduces theincidence of GMH (Ballabh et al., 2007), but angiogenic suppression inpremature infants would be undesirable, since it could impair lungmaturation (Thebaud, 2007).

Novel treatment strategies are desperately needed to combat GMH. Blockof SUR1 using glibenclamide is such a treatment, in particular aspectsof the invention. Glibenclamide pretreatment in humans is associatedwith significantly better outcomes from stroke (Simard et al., 2008;Kunte et al., 2007), and constant infusion of drug at doses below thosethat give hypoglycemia is highly effective in preventing progressivesecondary hemorrhage in the CNS (Simard et al., 2007). The presentexample is consistent with the embodiment that the SUR1-regulatedNC_(Ca-ATP) channel is causally linked to GMH. In particular embodimentsof the invention, glibenclamide and other compounds that block theexpression and/or activity of the channel are useful in reducing theincidence of this devastating complication of prematurity.

Example 15 Traumatic Brain Injury Embodiments

Traumatic brain injury (TBI) causes deficits in motor, sensory,cognitive, and emotional functions. This debilitating neurologicaldisorder is common in young adults and often requires life-longrehabilitation. A contusion injury to the brain is typically aggravatedby secondary injury, resulting in expansion of the original lesion andconcomitant worsening of neurological outcome. Mechanisms of secondaryinjury are diverse and may include cytotoxic processes, such asexcitotoxicity, free radical damage, apoptosis, inflammation, etc. Inaddition, secondary injury may result from microvascular dysfunction,including ischemia, edema, and “progressive secondary hemorrhage”, aphenomenon wherein capillaries gradually loose their structuralintegrity and become fragmented, resulting in extravasation of blood andformation of petechial hemorrhages. Whereas historically, ischemia andedema have been targeted for treatment, progressive secondary hemorrhagehas not, simply because hemorrhage has not been viewed as beingpreventable. However, blood is extremely toxic to neural tissues, as itincites free radical formation and inflammatory responses that areespecially damaging to myelin of white matter tracks, thereby worseningthe overall neurological injury. Thus, if secondary injury is to beminimized, it is important that progressive secondary hemorrhage bereduced.

The inventor has discovered that the novel ion channel, theSUR1-regulated NC_(Ca-ATP) channel is highly relevant to understandingsecondary injury in TBI (Simard et al., 2008). This channel is notconstitutively expressed, but is expressed only after injury to the CNS,with expression being particularly prominent in endothelial cells ofpenumbral capillaries surrounding the primary injury site (Simard etal., 2007). Originally, the work indicated that an ischemic/hypoxicinsult was required for de novo expression (Simard et al., 2006), butrecently, evidence was obtained that this channel is also newlyexpressed following trauma to the spinal cord (Simard et al., 2007) andbrain (see below).

The NC_(Ca-ATP) channel is unique (Simard et al., 2008). It conveysmonovalent but not divalent cations, it requires intracellular Ca²⁺, andchannel opening is triggered by depletion of intracellular ATP. Whenopened, the channel depolarizes the cell due to influx of Na⁺, drawingin Cl⁻ and water, leading to oncotic cell swelling and oncotic celldeath. When capillary endothelial cells undergo oncotic death, thestructural integrity of capillaries is lost, resulting in formation ofpetechial hemorrhages. Of particular importance, this channel isregulated by sulfonylurea receptor 1 (SUR1), just like pancreaticK_(ATP) channels. Unlike K_(ATP) channels, whose opening leads tohyperpolarization, opening of NC_(Ca-ATP) channels leads to celldepolarization. Opening of NC_(Ca-ATP) channels is prevented by thesulfonylurea, glibenclamide (glyburide), which protects cells thatexpress the channel from oncotic swelling and oncotic death. In rodentmodels of stroke and spinal cord injury, systemic administration oflow-dose glibenclamide is highly neuroprotective (Simard et al., 2006;2007; 2008). In human diabetics who coincidentally are takingsulfonylureas at the time of stroke, outcomes are highly favorablecompared to matched controls (Kunte et al., 2007).

The inventor has obtained experimental data that indicate that: (i)progressive secondary hemorrhage is prominent following percussion-TBI,with hemorrhage doubling during the first 12-24 hr; (ii) SUR1, theregulatory subunit of the channel, and TRPM4, the pore forming subunitof the channel, are abundantly upregulated post-TBI; (iii) progressivesecondary hemorrhage can be significantly reduced by low-doseglibenclamide; (iv) glibenclamide-treatment is associated withsignificant neurological and neurobehavioral functional improvement.Thus, in certain embodiments of the invention, glibenclamide, forexample, is useful for preventing, ameliorating, and/or treating TBI.

In one embodiment, there is established a useful treatment to reducesecondary injury related to microvascular dysfunction post-TBI. Sinceglibenclamide (glyburide) is a safe drug that has been used for over twodecades to treat type 2 diabetes in humans, providing treatment of TBIin humans that is critical to reducing secondary injury and thereforeoptimizing rehabilitation post-TBI.

In a specific embodiment as may be demonstrated in a rodent model ofTBI, properly timed treatment with the proper dose of the SUR1antagonist, glibenclamide, is believed to (i) minimize secondary injury(formation of edema and secondary hemorrhage); (ii) minimize lesionsize, limiting it to the original site of primary injury; and/or (iii)optimize neurofunctional, cognitive and psychophysiological recovery. Inanother specific embodiment, the time-course is determined forupregulation of the glibenclamide-sensitive, SUR1-regulated NC_(Ca-ATP)channel following percussion-TBI. In an additional specific embodiment,the time-window and optimal dose for treatment with glibenclamide isdetermined.

In an additional embodiment, the therapeutic efficacy is determined ofglibenclamide in male and female rats using a comprehensive battery ofneurofunctional, cognitive and psychophysiological tests assessed up to6 months post-TBI, for example.

TBI—the Clinical Problem

Each year, 1.5 million Americans sustain TBI. As a result of theseinjuries, 50,000 people die, 230,000 people are hospitalized andsurvive, and 80,000-90,000 people experience the onset of long-termdisability (Langlois et al., 2006; Thurman et al., 1999). TBI is theleading cause of death and disability in children and adults ages 1-44years. As detailed above, warfighters and veterans are also highly proneto suffer from TBI and its aftereffects (Warden, 2006; Sayer et al.,2008). Overall, more than 5 million Americans—2% of the U.S.population—currently live with disabilities resulting from TBI. Theconsequences in terms of physical impairments, functional limitations,disabilities, societal restrictions, and economic impact are practicallyimmeasurable.

In spite of its importance to civilian and military personnel, there isno effective therapy in clinical use that is specifically directedtowards ameliorating secondary brain injury after trauma. An importantreason for this unfortunate deficiency in clinical care is an incompleteunderstanding of cellular and molecular processes that underliesecondary brain injury. One important area of deficiency concernsmechanisms of secondary injury related to microvascular dysfunction, inparticular, progressive secondary hemorrhage.

TBI—Secondary Injury and Progressive Secondary Hemorrhage (PSH)

The pathophysiology of TBI is complex and involves multiple injurymechanisms that are spatially and temporally specific, including bothprimary and secondary injury mechanisms. A consistent pattern ofcytotoxic and microvascular abnormalities can be documented in the earlyposttraumatic period (Dietrich et al., 1994) with many secondary injurymechanisms remaining active for days to weeks after the primary insult.It is believed that by successfully targeting one or more mechanism ofsecondary injury, the burden of injury will be lessened, rehabilitationwill be more successful, and the overall outcome will improve pursuantto the treatments and methods disclosed herein.

Numerous mechanisms of secondary injury have been identified, includingcytotoxic mechanisms involving excitotoxicity, free radical production,apoptosis, inflammation and others, as well as microvascularabnormalities responsible for ischemia and edema (Bramlett and Dietrich,2007; Raghupathi, 2004). Notably, one pathophysiological process that islargely unrecognized as a mechanism of secondary injury is “progressivesecondary hemorrhage” (PSH). Contusion of brain often results information of intraparenchymal petechial hemorrhages (Dietrich et al.,1994; Cortez et al., 1898; Oertel et al., 2002; Schmidt and Grady,1993). Formation of petechial hemorrhages has been associated with smallvenules (Dietrich et al., 1994), but less well appreciated is the factthat hemorrhages are frequently complicated by “blossoming” or expansion(Cortez et al., 1989; Oertel et al., 2002; Vajtr et al., 2008). Althoughsometimes erroneously attributed to continued bleeding of vesselsfractured by the original trauma, this phenomenon actually represents asecondary pathological process, as we have shown in spinal cord injury(Simard et al., 2007). PSH occurs during the first several hours after atraumatic insult. It results from progressive catastrophic failure ofthe structural integrity of capillaries, and is characterized byformation of small discrete satellite (petechial) hemorrhages in tissuessurrounding the site of primary injury. With time, petechial hemorrhagesincrease in number and eventually coalesce into a hemorrhagic lesionthat encompasses the entire site of primary injury. PSH is particularlydamaging because it greatly expands the volume of neural tissuedestroyed by the primary injury. The capillary dysfunction implicit withPSH causes tissue ischemia and hypoxia, and the hemorrhage thatcharacterizes PSH is exquisitely toxic to CNS cells (Regan and Guo,1998; Wang et al., 2002), further injuring neural tissues due tooxidative stress and inflammation. Together, these processes render PSHthe most destructive mechanism of secondary injury involving the CNS.

Two molecular mechanisms can potentially account for PSH: (i)upregulation of matrix metalloproteinases (Vajtr et al., 2008; Vilaltaet al., 2008), (ii) upregulation of the capillary endothelialSUR1-regulated NC_(Ca-ATP) channel (see below and Simard et al., 2007).Both occur post-TBI. In general, research has identified variouspromising pharmacological compounds that specifically antagonize many ofthe commonly identified secondary mechanisms of injury that contributeto TBI. However, none explicitly targets PSH post-TBI. In certainaspects, the role of SUR1-regulated NC_(Ca-ATP) channels is evaluated inPSH post-TBI it is believed that glibenclamide has utility in reducingor eliminating PSH post-TBI.

The SUR1-Regulated NC_(Ca-ATP) Channel

Channel properties. The properties of the SUR1-regulated NC_(Ca-ATP)channel have been reviewed (Simard et al., 2007; Simard et al., 2008;Simard et al., 2007). It is a 35 pS cation channel that conductsinorganic monovalent cations, but is impermeable to Ca²⁺ and Mg²⁺ (Chenand Simard, 2001). Channel opening requires nanomolar concentrations ofCa²⁺ on the cytoplasmic side, and is blocked by intracellular ATP (EC₅₀,0.79 μM). Like K_(ATP) channels, SUR1-regulated NC_(Ca-ATP) channels areblocked by first and second generation sulfonylureas, tolbutamide (EC₅₀,16.1 μM) and glibenclamide (EC₅₀, 48 nM) (Chen et al., 2003). Recentwork has shown that the pore-forming subunit of the channel is TRPM4(see below), (Simard et al., 2007), but at present, no high affinity,high specificity drugs are available to block TRPM4.

Channel expression. The SUR1-regulated NC_(Ca-ATP) channel is notconstitutively expressed, but is expressed in the CNS under conditionsof injury or hypoxia. The channel was first discovered in reactiveastrocytes obtained from the hypoxic inner zone of the gliotic capsulepost-stab injury and foreign body implantation (Chen et al., 2001; Chenet al., 2003). Since then, it has been identified using patch clampelectrophysiology in neurons from the core of an ischemic stroke (Simardet al., 2006) and in cultured human and mouse endothelial cellssubjected to hypoxia (Simard et al., 2007).

Apart from patch clamp recordings to demonstrate presence of thechannel, CNS tissues have been analyzed to detect the regulatory subunitof the channel, SUR1, at protein and mRNA levels. Normally, SUR1 isexpressed in some neurons, but not in astrocytes or capillaries.Post-injury, SUR1 is strongly upregulated in several rodent models ofCNS injury, including models of cerebral ischemia (Simard et al., 2006),penetrating brain injury with foreign body (Chen et al., 2003), and SCI(Simard et al., 2007). Upregulation of SUR1 is found in all members ofthe neurovascular unit, i.e., neurons, astrocytes and capillaryendothelial cells.

Channel function. The consequences of opening the SUR1-regulatedNC_(Ca-ATP) channel have been studied in cells by depleting ATP to mimicinjury conditions. ATP depletion induces a strong inward current thatdepolarizes the cell completely to 0 mV. Cells subsequently undergooncotic cell swelling (cytotoxic edema). Eventually, ATP-depletion leadsto cell death, predominantly by non-apoptotic, propidium iodide-positiveoncotic (necrotic) cell death, which can be blocked by glibenclamide(Simard et al., 2006).

Glibenclamide Block of SUR1—In Vivo Models of CNS Injury

The effect of glibenclamide was studied in rodent models of ischemicstroke. In a model of malignant cerebral edema, glibenclamide reducedmortality and cerebral edema (excess water) by half (Simard et al.,2006). In a model of stroke induced by thromboemboli, glibenclamidereduced lesion volume by half, and its use was associated with corticalsparing that was attributed to improved leptomeningeal collateral bloodflow due to reduced mass effect from edema (Simard et al., 2006).

The effect of glibenclamide was studied in a rodent model of spinal cordinjury (SCI) (Simard et al., 2007). Acutely, SCI results in progressivesecondary hemorrhage, characterized by a progressively expansive lesionwith fragmentation of capillaries, hemorrhage that doubles in volumeover 12 hr, tissue necrosis and severe neurological dysfunction.Necrotic lesions are surrounded by widespread upregulation of SUR1 incapillaries and neurons. Following SCI, block of SUR1 by glibenclamideessentially eliminates capillary fragmentation and progressive secondaryhemorrhage, is associated with a 3-fold reduction in lesion volume, andresults in marked neurobehavioral functional improvement.

Role of the channel in edema and hemorrhage. Edema and progressivesecondary hemorrhage are key mechanisms of secondary injury post-TBI(Marmarou, 2007; Unterberg et al., 2004). Edema resulting from TBI orischemia can lead to raised ICP and brain herniation. Early progressivehemorrhage occurs in almost 50% of head-injured patients, usuallyfollowing contusion injury, and it too is associated with elevations inICP (Oertel et al., 2002; Smith et al., 2007; Xi et al., 2006).

Molecular mechanisms involved in cerebral ischemia, including cytotoxicedema, vasogenic edema, and hemorrhagic conversion were recentlyreviewed (Simard et al., 2007). Although mechanisms are complex and notcompletely understood, evidence has accumulated that SUR1-regulatedNC_(Ca-ATP) channels play a critical role in each of these, and thatblock of the channel by glibenclamide yields significant beneficialeffects. To date, most of the work has focused on brain ischemia andSCI, but strong data presented below indicate that the same mechanismsare at play in TBI.

Glibenclamide—Benefit in Human Stroke

An outcome analysis was carried out of patients with diabetes mellitus(DM) hospitalized within 24 hr of onset of acute ischemic stroke in theNeurology Clinic, Charite Hospital, Berlin, Germany, during 1994-2000(Kunte et al., 2007). After exclusions, the cohort comprised 33 patientstaking a sulfonylurea (e.g., glibenclamide) at admission throughdischarge (treatment group) and 28 patients not on a sulfonylurea(control group). The primary outcome was a decrease in NationalInstitutes of Health Stroke Scale (NIHSS) of 4 points or more fromadmission to discharge or a discharge NIHSS score=0, which is considereda “major neurological improvement”. The secondary outcome was adischarge modified Rankin Scale (mRS) score of 2 or less, whichsignifies functional independence. The primary outcome was reached by36% of patients in the treatment group and 7% in the control group (oddsratio=7.5 in favor of sulfonylurea; P=0.007). The secondary outcome wasreached by 81.8% vs. 57.1% (odds ratio=3.4 in favor of sulfonylurea;P=0.035).

In particular embodiments of the invention, secondary hemorrhage andlesion expansion that develops over time following percussion-TBI can beprevented by blocking NC_(Ca-ATP) channels with glibenclamide, and thatby doing so, a substantial improvement in neurofunctional outcome can beachieved.

Work on Rodent Model of Percussion-TBI

The model of percussion-TBI. The percussion-TBI model that has beenstudied is an exemplary gravity-driven, parasagittal mechanicalpercussion model similar to the gravity-driven, parasagittal fluidpercussion model (Thompson et al., 2005; Fujimoto et al., 2004), exceptthat the impact force is transmitted via a blunt mechanical impactorinstead of a fluid column. Unlike typical weight drop devices thatutilize a small diameter impactor head with restricted penetration(Bullock et al., 1995; Suh et al., 2000) in the model used by theinventor, TBI is created with an impactor rod tipped with a 5-mm Teflonball (4 gm total) activated by vertical weight drop. Like fluidpercussion, the model has unrestricted penetration, disperses the forceover an area of ˜20 mm² and transiently displaces a larger volume ofbrain tissue than a small diameter impactor with restricted penetration.

Young adult male Long-Evans rats, 240-280 gm, were studied. Rats wereanesthetized (Ketamine and Xylazine) and physiological parametersincluding temperature and blood gases were maintained within appropriatephysiological ranges. With the head fixed in a stereotaxic frame, a 6-mmcircular craniectomy was created abutting the sagittal and lambdoidalsutures. A posterior location was chosen to emphasize damage tounderlying hippocampus (Vink et al., 2001; Floyd et al., 2002). Theimpactor was activated using a 10-gm weight dropped from 10 cm, whichproduced a transient impact pressure of 2.5-3 atm (FIG. 13). Shamcontrols underwent craniectomy without percussion.

For some studies, the effect of treatment with glibenclamide wasassessed. Immediately after TBI, rats were implanted with mini-osmoticpumps (Alzet 2002, 0.5 ml/hr; Durect Corporation, Cupertino, Calif.)that delivered either vehicle (DMSO/saline) or drug (glibenclamide,Sigma, in DMSO/saline) subcutaneously (Simard et al., 2006; Simard etal., 2007). Pharmacokinetic analysis indicated that 3 hr were requiredto achieve 90% steady-state serum drug levels. The dose of glibenclamidedelivered was 200 ng/hr, which at 3 hr, resulted in a non-significantdecrease in serum glucose, from 236±15 to 201±20 (5-6 rats per group;p=0.19). The dose of DMSO delivered was 40 nl/hr, which is 300 timesless than that associated with neuroprotection.

Mortality, pathology and behavior. The acute-stage outcome (24 hr)produced in our percussion model with 2.5-3 atm transient pressure wassimilar to reports with fluid percussion of 2.5-3 atm (Thompson et al.,2005; Fujimoto et al., 2004; Dixon et al., 1987). The mortality of 15%was similar (Dixon et al., 1987). As with fluid percussion, a combinedfocal and diffuse injury was produced. A hemorrhagic contusion wasapparent at the site of percussion that extended below the corpuscallosum to involve much of the ipsilateral hippocampus and deeperstructures (FIGS. 14A-14B, and 15A-15E). There was significant cell andtissue loss in hippocampal CA2/CA3 and hilus ipsilateral to the injurysite (see FIGS. 19A,19C). Evidence of contralateral injury was also seen(FIG. 15B). Compared to sham controls, survivors exhibited markedreduction in spontaneous movements, in startle response, in exploratorymovements in open field testing and much less frequent verticalexploration in an open cylinder test (see FIG. 20).

SUR1 is upregulated in rats post-TBI. Rats were studied for SUR1expression. Montages of sections immunolabeled at 3 hr showed littleSUR1, but by 24 hr, SUR1 was prominent both ipsilaterally andcontralaterally (FIGS. 15A,15B). Co-immunolabeled sections showed thatnewly expressed SUR1 co-localized with NeuN (neurons; not shown) andwith vonWillebrand factor or vimentin (capillaries; FIGS. 15C,15D).Upregulation was confirmed with Western blots (FIG. 15E).

SUR1 is upregulated in humans post-TBI. To ascertain the relevance ofthese observations to humans, we also studied SUR1 expression in biopsyspecimens from patients who required craniotomy fordebridement/decompression 6-30 hr post-insult. Immunohistochemistry forSUR1 and in situ hybridization for Abcc8, which encodes SUR1, showedprominent upregulation in neurons and microvessels in 2/2 patientsstudied with gunshot wound to the brain (FIGS. 16A-16F) and in onepatient with intracerebral hematoma due to rupture of arteriovenousmalformation (see Simard et al., 2008). This is consistent with themethods and treatments disclosed herein, and supports the use of SUR1antagonists in the treatment of human TBI patients.

In rat, progressive secondary hemorrhage manifests as an increase inextravasated blood. Using the model of percussion-TBI, data was obtainedshowing that the magnitude of the hemorrhage into the brain increasedprogressively over the first 24 hr after injury. Animals were sacrificedat ½, 6 and 24 hr after percussion-TBI (n=3-5 rats per group). They wereperfused with heparinized saline to remove intravascular blood andportions of brain encompassing the lesion were homogenized and processedusing Drabkin's reagent to convert hemoglobin to cyanomethemoglobin forspectrophotometric measurements (Simard et al., 2007). Values roseprogressively over the first 24 hr, reaching half-maximum 5.2 hrpost-injury, and maximizing only ˜10 hr post-injury (FIGS. 17A-17C). Thefact that secondary hemorrhage is progressive over such a long period oftime is seldom appreciated, but forms an underlying rationale fordirectly attacking this severely harmful cause of secondary injurypost-TBI.

Block of SUR1 with glibenclamide reduces progressive secondaryhemorrhage. We assessed the effect of glibenclamide on progressivesecondary hemorrhage. As above, animals were sacrificed at ½, 6 and 24hr after percussion-TBI. Glibenclamide treatment did not affect thevolume of blood measured/2 hr post-injury, indicated a comparablemagnitude of injury between groups (FIGS. 17A-17C). However,glibenclamide prevented further increases in blood that were observed atlater times in vehicle-treated controls (FIGS. 17A-17C). At 24 hrpost-injury, tissue homogenates from glibenclamide-treated animals werevisibly less bloody that those from vehicle-treated animals (FIGS.17A-17C, insert). Overall, these data indicate that glibenclamide washighly effective in reducing progressive secondary hemorrhage post-TBI.

Glibenclamide effect on secondary hemorrhage is not due to an effect oncoagulation or to inhibition of MMP. In uninjured rats given the samedose as above, glibenclamide had no effect on tail bleeding time(19.3±1.9 vs. 21.5±3.1 sec; n=3-5; P=0.6).

In stroke, hemorrhagic conversion has been attributed to activation ofmatrix metalloproteinases (MMP) (Justicia et al., 2003; Lorenzl et al.,2003; Romanic et al., 1998). It was assessed whether glibenclamide mightbe directly inhibiting MMPs. Zymography of recombinant MMPs showed thatgelatinase activity assayed in the presence of glibenclamide was thesame as that assayed without it, although gelatinase activity wasstrongly inhibited by commercially available MMP inhibitor II (FIG. 18).This finding makes it unlikely that glibenclamide was acting directlyvia MMP inhibition to decrease secondary hemorrhage post-TBI, andindicated instead that a mechanism involving SUR1-regulated NC_(Ca-ATP)channels in capillary endothelium was likely to be involved, as we haveshown recently for SCI (Simard et al., 2007).

Block of SUR1 with glibenclamide reduces lesion size and spareshippocampal neurons. The beneficial effect of glibenclamide onprogressive secondary hemorrhage was associated with a reduction inlesion area on coronal sections at the epicenter of injury, from 8.2±1.3to 4.4±0.8 mm² (10 rats/group; P=0.025), at 7 days post-TBI (FIG. 19Aversus FIG. 19B).

Niss1 stained sections also showed that glibenclamide treatment wasassociated with sparing of hippocampus, including sparing of neurons inCA1, CA3 and dentate gyrus regions (FIGS. 19A-19D). Neuronal loss,pyknotic cells and hemorrhages observed in vehicle treated controls weremuch less likely to be seen with glibenclamide treatment (FIGS.19A-19D).

Block of SUR1 with glibenclamide improves neurobehavioral function. Thedata included only simple testing of neurobehavioral function.Spontaneous forelimb use (SFU) was quantified and spontaneous verticalexploration (SVE) was quantified during 7 days post-TBI. SFU measuressensorimotor asymmetry (Schallert et al., 2000) whereas SVE measures notonly vestibulomotor function but also time spent in exploratoryactivity. At 2 days post-TBI, glibenclamide treatment was associatedwith an increase in spontaneous use of the forelimb contralateral to theinjury from 3.5±3.5% in controls to 16.5±3.4% in the treatment group(P=0.05). At 1, 2 and 7 days post-TBI, glibenclamide-treated ratsconsistently exhibited significantly greater SVE scores than controls(FIG. 20).

Transient receptor potential M4 (TRPM4) pores physically associates withSUR1 and is upregulated in penumbral capillaries post-TBI. TheSUR1-regulated NC_(Ca-ATP) channel is composed of molecularly distinctregulatory and pore-forming subunits encoded by different genes. SUR1was previously identified as the regulatory subunit (Simard et al.,2006; Chen et al., 2003) and it is considered that TRPM4 forms thepore-forming subunit, based on essentially identical biophysicalproperties of NC_(Ca-ATP) and TRPM4 channels (Simard et al., 2007).Co-immunoprecipitation studies were carried out to examine the physicalassociation between SUR1 and TRPM4. Western blots showed that totallysate from injured tissue exhibited abundant TRPM4 protein (FIG. 21,middle lane), and that immunoprecipitation using anti-SUR1 antibodyyielded a product also identified as TRPM4 (FIG. 21, right lane),confirming physical association between SUR1 and TRPM4. Moreover, aswith SUR1, TRPM4 is abundantly upregulated especially in penumbralcapillaries post-TBI (FIGS. 22A-22C). In certain aspects, the temporalprofile for SUR1 and TRPM4 mRNA and protein expression post-TBI isdetermined.

Studies on isolation of brain microvascular complexes and patch clamp ofcapillaries. Microvascular complexes were isolated from normal(uninjured) rat brain using a method based on perfusion with magneticparticles (details of method given below). Magnetic separation yieldedmicrovascular complexes that typically included a precapillary arterioleplus attached capillaries (FIGS. 23A-23C). As is evident from the image,unambiguous identification of capillaries for precise positioning of thepipette for patch clamping attached capillary endothelial cells isreadily achievable (FIG. 23A, arrows).

Capillary endothelial cells still attached to intact microvascularcomplexes were patch clamped using a conventional whole cell method.Cells were studied with standard physiological solutions in the bath andin the pipette, including 2 mM ATP in the pipette solution. Membranecurrents showed time-dependent activation (FIG. 23B) with a weaklyrectifying current-voltage (I-V) relationship that reversed near −50 mV(FIG. 23C). These recordings demonstrate the feasibility of patchclamping freshly isolated capillary endothelial cells still attached tointact microvascular complexes from brain.

In certain embodiments of the invention, SUR1, which regulates the novelNC_(Ca-ATP) channel, is directly responsible for critical pathologicalmechanisms of secondary injury, most importantly, progressive secondaryhemorrhage, and that by blocking this channel with the highly potent andsafe antagonist, glibenclamide (glyburide), significant improvements inoutcome can be obtained post-TBI. Demonstrating these concepts advancespharmaceutical treatments that greatly improves management of TBI andimproves existing strategies for rehabilitation. Modern techniques ofmolecular biology, electrophysiology and neurobehavioral may beemployed, for example.

In one case, the time course for upregulation of the molecularcomponents of the channel as well as of functional channels, which isrequired to define the time-window for treatment, is determined. Inanother case, one can evaluate the effect of channel inhibition on edemaand hemorrhage using various doses of glibenclamide beginning at varioustimes post-injury, to determine the allowable time-window and theoptimal dose for treatment. Finally, in an additional case, one canconfirm the therapeutic efficacy of glibenclamide in male and femalerats using a comprehensive battery of neurofunctional, cognitive andpsychophysiological tests assessed up to 6 months post-TBI.

The model of percussion-TBI. In certain aspects data were obtained usinga mechanical percussion device that was designed and built, whichproduced injury forces (see FIG. 13) and yielded brain damage (see FIGS.14A and 14B) comparable to moderate-to-severe fluid percussion (Thompsonet al., 2005). Although the device yielded quite reproducible results(FIGS. 14A, 14B, 17A, 19A are from 4 different rats), fluid percussioninjury (FPI) has long been used and is widely accepted in TBI research(Thompson et al., 2005). Although some injury parameters are bettercontrolled using a controlled cortical impact (CCI) device rather than aFPI device, FPI is preferred over CCI, in certain cases, because CCIgenerally produces a more focused injury compared with FPI and overall,TBI is less severe with CCI compared to FPI (Obenaus et al., 2007).Injuries produced by parasagittal FPI are more diffuse and, importantly,are more likely to involve hippocampus. These differences inevitablyhave implications with respect to behavioral and functional outcomes(Fujimoto et al., 2004; Cernak, 2005).

Thus, a fluid percussion model, with a percussion pressure of ˜3 atm maybe used in studies as disclosed herein. Controls undergo sham surgery(craniectomy without percussion). Young adult (12 weeks) male (Objective1-3) or female (Objective 3) Long-Evans rats are suitable animals foruse in the studies disclosed herein.

Drug treatment following TBI. Typically, studies of drug interventionspost-TBI utilize one or more injections of drug during the post-injuryperiod. This technique yields plasma levels of drug that can fluctuatewidely between peaks and troughs, depending on (usually unknown)pharmacokinetic parameters. A constant infusion of drug is utilized,with the aim of achieving constant occupancy of high-affinity receptorswithout potential complications inherent with transiently excessive druglevels. Thus, within 2-3 min of injury, mini-osmotic pumps (Alzet) areimplanted over the dorsal thorax to deliver either vehicle or drugsubcutaneously, with pumps fitted with “Lynch coils” to obtain anydesired delay in start of treatment. This technique has beensuccessfully employed in previous studies (Simard et al., 2006; Simardet al., 2007).

For certain studies, glibenclamide was delivered at 200 ng/hr (noloading dose). For other studies, the effects of various doses ofglibenclamide, including use of a loading dose, are characterized. Thepurpose is to mimic treatment that would be implemented in humans,including use of a loading dose and constant infusion, coupled with adelay in start of treatment. (One case use i.p. and s.q. routes in ratsinstead of i.v., as would be used in humans, for example.)

In certain embodiments of the invention, SUR1-regulated NC_(Ca-ATP)channels are upregulated in neurons and capillary endothelial cells overseveral hours after TBI Previous work identified SUR1 as the regulatorysubunit of the NC_(Ca-ATP) channel (Simard et al., 2006; Simard et al.,2007; Chen et al., 2003). New work has identified transient receptorpotential melastatin 4 (TRPM4) as the pore forming subunit. Thus,determining the time course for channel upregulation post-TBI employsstudying expression of mRNA and protein for these two molecularcomponents, in certain cases. However, expression of subunits does notnecessarily assure expression of pathologically functional channels.Therefore, full characterization of the time course of channelexpression also utilizes patch clamp experiments to document theexpression of functional channels in capillary endothelial cells andneurons.

Specific embodiments on percussion-TBI indicate that SUR1 protein isupregulated 24 hr after injury in capillaries and neurons. However, thebeneficial effect of glibenclamide on progressive secondary hemorrhageat 6 hr (FIGS. 17A-17C) indicates that channels are upregulated muchearlier than 24 hr. Indeed, previous work in stroke indicated that SUR1itself, as well as functional SUR1-regulated NC_(Ca-ATP) channels areupregulated in neurons as early as 2-3 hr after onset of ischemia(Simard et al., 2006). Channel upregulation in neurons and astrocytes isthought to be critical for cytotoxic edema, whereas channel upregulationin capillary endothelial cells is thought to be critical for ionicedema, vasogenic edema and hemorrhagic conversion (Simard et al., 2007).Understanding the time course for channel expression in different celltypes is crucial for determining the treatment window for glibenclamide.

Overview of Studies

In certain cases, the time course for upregulation of NC_(Ca-ATP)channels following percussion-TBI is determined. This utilizes threeexemplary series of studies. First, Western blots are used to measurethe increase in SUR1 and TRPM4 protein and qPCR is used to measure theincrease in mRNA for SUR1 and TRPM4. The qPCR experiments provide directconfirmation of involvement of transcription, and also indirectlyvalidate the Western blot studies. As regards specificity of antibody,it was previously shown that the anti-SUR1 antibody to be used forWesterns (and immunochemistry, see below) exhibits a high degree ofspecificity for SUR1, and labels only a single band (180 kDa) in therange between 116-220 kDa (simard et al., 2006). Secondly, it isdetermined which cells are actually upregulating transcriptionalexpression of SUR1 and TRPM4. This is done using double immunolabelingexperiments, with validation provided at the mRNA level using in situhybridization. Third, it is determined whether newly upregulated SUR1and TRPM4 are associated with functional NC_(Ca-ATP) channels, whichemploys patch clamp experiments.

Experimental Design:

Time-course for SUR1 and TRPM4 protein and mRNA, using Westerns and qPCR

SUR1 and TRPM4 protein is measured in 7 groups of animals: in controls(sham surgery) and in animals with ˜3 atm percussion-TBI at 6 timesafter injury, at ¾, 1.5, 3, 6 12, 24 hr. Blots are stripped andre-blotted for Kir6.1 and Kir6.2, to show non-involvement of K_(ATP), aspreviously (Simard et al., 2006). Each of the seven groups requires 3rats per group.

SUR1 and TRPM4 mRNA are measured in 7 groups of animals: in controls(sham surgery) and in animals with ˜3 atm percussion-TBI at 6 timesafter injury, at %, 1.5, 3, 6 12, 24 hr. Each of the seven groupsrequire 3 rats per group. (NB: separate groups are required for proteinand mRNA because tissues are processed differently)

Specific Methods:

Preparation of tissues. After death, animals are perfused withheparinized saline to remove blood from the intravascular compartment.For the qPCR experiments, the perfusion solution includes RNA later(Ambion, Auston Tex.), to prevent RNA degradation and optimizequantification. The injured left hemisphere is sectioned to include 5 mmrostral and 5 mm caudal to the impact site (2×impact diameter), withsampling including parietal lobe and underlying tissues, includinghippocampus. Harvested tissues are flash frozen in liquid nitrogen andstored at −80° C. until processed.

Western blots. Lysates of whole tissues are prepared by homogenizing inRIPA lysis buffer, and electrophoretic gels (NuPAGE® 3-8% Tris-Acetategels; Novex, Invitrogen, Carlsbad, Calif.) are processed as described(Perillan et al., 2002). Blots are analyzed for SUR1 (SC-5789; SantaCruz Biotechnology), TRPM4 (SC-27540; Santa Cruz), Kir6.1 or Kir6.2(Santa Cruz). Membranes are stripped and re-blotted for β-actin (1:5000;Sigma), which is used as loading control. Detection is carried out usingthe ECL system (Amersham BioTBIences, Inc.) with routine imaging (FujiLAS-3000) and quantification (Scion Image, Scion Corp, Frederick, Md.).

The specificity of the SUR1 antibody has been documented (Simard et al.,2006). The specificity of the Kir6.x antibodies is confirmed withWestern blots on insulinoma RIN-m5f cells (Kir6.2) and rat heart(Kir6.1). The specificity of the TRPM4 antibody using TRPM4heterologously expressed in COS-7 cells is confirmed.

qPCR. Lysates of whole tissues are prepared by homogenizing in RNA lysisbuffer (Promega). There is reverse transcription of 1 μg of total RNA(normalized conditions) with random hexonucleotides according to themanufacturer's protocol (Applied Biosystems) and real-time PCR reactionswith an ABI PRISM 7300 Sequence Detector System (Applied Biosystems) areperformed using a TaqMan based protocol in a 96-well plate format. TaqMan probes and primers are selected with Primer Express 2.0 (AppliedBiosystems) software and synthesized by Applied Biosystems. Primersequences: H1 histone family member (housekeeping gene):CGGACCACCCCAAGTATTCA (forward) (SEQ ID NO:5); GCCGGCACGGTTCTTCT(reverse) (SEQ ID NO:6); CATGATCGTGGCTGCTATCCAGGCA (SEQ ID NO:7) (TaqManProbe). rSUR1(NM_013039.1): GAGTCGGACTTCTCGCCCT (forward) (SEQ ID NO:8);CCTTGACAGTGGACCGAACC (reverse) (SEQ ID NO:9); TTCCACATCCTGGTCACACCGCTGT(SEQ ID NO:10) (TaqMan Probe); rTRPM4 (XM_574447): AGTTGAGTTCCCCCTGGACT(forward) (SEQ ID NO:11); AATTCCAGTCCCTCCCACTC (reverse) (SEQ ID NO:12).Amplification reactions are performed using a TaqMan amplification kit(Applied Biosystems) according to the manufacturer's protocol, in 25 μlof reaction volume with 2 μl of cDNA. The amplification program consistsof a 5-min holding period at 95° C., followed by 40 cycles of 95° C. for30 sec, 60° C. for 30 sec and 72° C. for 30 sec. Relative quantificationis performed using a standard curve method (User Bulletin #2, PE AppliedBiosystems). All samples are run in triplicate.

Statistical analysis: Means will be compared using ANOVA.

Cellular Localization, Using Immunohistochemistry and In SituHybridization, for SUR1 and TRPM4.

In these studies, SUR1 and TRPM4 are the focus, but now with the intentof determining the cell types responsible for SUR1 and TRPM4upregulation. For this, one can perform double immunolabelingexperiments, labeling neurons with NeuN, astrocytes with GFAP, andcapillary endothelial cells with vonWillebrand factor and vimentin(Schnittler et al., 1998). Also, one can perform in situ hybridizationexperiments to further validate the SUR1 and TRPM4 immunohistochemistry.

Immunolabeling is performed for SUR1 and TRPM4 plus double labeling fora cell-specific marker (NeuN, GFAP, vimentin, vWf) in 7 groups ofanimals: in controls (sham surgery) and in animals with ˜3 atmpercussion-TBI at 6 times after injury, at %, 1.5, 3, 6 12, 24 hr. Eachof the seven groups may include, for example, 3 animals/group.

Confirmatory in situ hybridization studies are performed for SUR1 mRNAin 4 groups of animals: in controls (sham surgery) and in animals with˜3 atm percussion-TBI at 3 times after injury, at 1.5, 6 and 24 hr.These studies can utilize tissues from the same rats as used forimmunolabeling.

Specific Methods:

Preparation of tissues. After death, animals are perfused withheparinized saline to remove blood from the intravascular compartmentfollowed by 4% paraformaldehyde. The brain is harvested, cut to include5 mm rostral and 5 mm caudal to the impact site. The brain iscryoprotected using 30% w/v sucrose.

Immunohistochemistry. Cryosections are used for double immunolabeling(SUR1+NeuN, SUR1+GFAP; SUR1+vWf) or (TRPM4+NeuN, TRPM4+GFAP; TRPM4+vWf),using standard techniques (Chen et al., 2003). After permeabilizing(0.3% Triton X-100 for 10 min), sections are blocked (2% donkey serumfor 1 hr; Sigma D-9663), then incubated with primary antibody directedagainst SUR1 (1:200; 1 hr at room temperature then 48 h at 4° C.;SC-5789; Santa Cruz Biotechnology) or TRPM4 (1:200 overnight at 4° C.;Santa Cruz). After washing, sections are incubated with fluorescentsecondary antibody (1:400; donkey anti-goat Alexa Fluor 555; MolecularProbes, OR). For co-labeling, one can use primary antibodies directedagainst NeuN (1:100; MAB377; Chemicon, CA); GFAP (1:500; CY3 conjugated;C-9205; Sigma, St. Louis, Mo.); vonWillebrand factor (1:200; F3520,Sigma) vimentin (1:200; CY3 conjugated; C-9060, Sigma) and, as needed,species-appropriate fluorescent secondary antibodies. Fluorescentsignals are visualized using epifluorescence microscopy (Nikon EclipseE1000).

In situ hybridization. Fresh-frozen sections are post-fixed in 5%formaldehyde for min. Digoxigenin-labeled probes (SUR1: antisense:′5-GCCCGGGCACCCTGCTGGCTCTGTGTGTCCTTCCGCGCCTGGGCATCG-3′ (SEQ ID NO:13);TRPM4: (antisense:′5-CCAGGGCAGGCCGCGAATGGAATTCCCGGATGAGGCTGTAGCGCTGCG-3′ (SEQ ID NO:14);GeneDetect)”) are designed and supplied by GeneDetect (Brandenton, Fla.)and hybridization is performed according to the manufacturer's protocol(Simard et al., 2006; Simard et al., 2007).

Channel Function Using Patch Clamp Electrophysiology on Isolated Cells

It is determined electrophysiologically whether upregulated SUR1 andTRPM4 subunits form functional NC_(Ca-ATP) channels in capillaryendothelial cells and neurons. The salient biophysical features of thechannel (Simard et al., 2008) include: (i) the channel conducts Cs⁺, sothat recordings with Cs⁺ as the only permeant cation unambiguouslydistinguish between SUR1-regulated NC_(Ca-ATP) channels andSUR1-regulated K_(ATP) channels; (ii) the channel is regulated by SUR1,so that block of a Cs⁺ conductance by low concentrations ofglibenclamide identifies the channel with virtual certainty.

The data on TBI indicate that glibenclamide is highly effective inreducing progressive secondary hemorrhage. In certain aspects, this highpotency reflects not only the high affinity of the drug at the receptor(EC₅₀=48 nM at neutral pH, 6 nM at pH 6.8) (Chen et al., 2003), but alsothe fact that ischemic or injured tissues are at lower pH (≈6.5), 42coupled with the relatively acidic pKa of glibenclamide (6.3), resultingin greater lipid solubility and thus greater tissue concentration of thecompound in ischemic regions. This is tested directly.

Cell isolation is performed twice weekly, with each batch of freshlyisolated cells studied over the course of 2 days, allowing patch clampexperiments ˜4 days/week.

Specific Methods:

Isolation of brain microvessels with attached capillaries. The methodused (see FIGS. 23A-23C) is adapted from Harder et al. (1994) Tissuesare prepared at 3-5 hr post-TBI. A rat undergoes transcardiac perfusionof 50 ml of heparinized PBS containing a 1% suspension of iron oxideparticles (10 μm; Aldrich Chemical Co.). The contused brain is removed,the pia and pial vessels are stripped away, the tissue is minced intopieces 1-2 mm3 with razor blades. Tissue pieces are incubated withdispase II (2.4 U/ml; Roche) for 30 min with agitation in the incubator.Tissues are dispersed by trituration with a fire-polished Pasteurpipette. Microvessels are adhered to the sides of 1.5 ml Eppendorf tubesby rocking 20 min adjacent to a magnet (Dynal MPC-S magnetic particleconcentrator; Dynal Biotech, Oslo, Norway). Isolated microvessels arewashed in PBS×2 to remove cellular debris and are stored at 4° C. inphysiological solution (Harder et al., 1994). For patch clamp study ofcapillary cells, an aliquote of microvessels is transferred to therecording chamber, and using phase contrast microscopy, capillaries nearthe end of the visualized microvascular tree are targeted for patchclamping.

Isolation of neurons. Neurons are isolated from vibratome cut brainsections as we described. 2 Tissues are prepared at 3-5 hr post-TBI. Thebrain is removed and vibratome sections (300 μm) are processed asdescribed (Hainsworth et al., 2001) to obtain single neurons for patchclamping. Selected portions of slices are incubated at 35° C. in HBSSbubbled with air. After 30 min, the pieces are transferred to HBSScontaining 1.5 mg/ml protease XIV (Sigma). After 30-40 min of proteasetreatment, the pieces are rinsed in enzyme-free HBSS and mechanicallytriturated. For controls, cells were utilized from sham animals. Cellsare allowed to settle in HBSS for 10-12 min in a plastic Petri dishmounted on the stage of an inverted microscope. Large and medium-sizedpyramidal-shaped neurons are selected for recordings.

Patch clamp electrophysiology. Numerous papers present detailed accountsof the patch clamp methodologies that may be use, including whole-cell,inside-out, outside-out and perforated patch methods (Chen et al., 2001;Chen et al., 2003; Perillan et al., 2002; Perillan et al., 1999;Perillan et al., 2000).

The overall design of the studies follows a strategy previously usedwith reactive astrocytes and neurons for characterizing the NC_(Ca-ATP)channel (Simard et al., 2006; Chen et al., 2001; Chen et al., 2003).Initial studies are carried out using a whole-cell perforated patchconfiguration to characterize macroscopic currents, and to test theoverall response to ATP depletion induced by exposure to themitochondrial poisons, Na azide or Na cyanide/2-deoxyglucose, as used inpreviously (Simard et al., 2006; Simard et al., 2007; Chen et al.,2001). This configuration is also useful for characterizing the responseto the SUR1 activators: if the cell expresses NC_(Ca-ATP) channels,diazoxide activates an inward current that reverses near zeromillivolts, whereas if the cell expresses K_(ATP) channels, diazoxideactivates an outward current that reverses near −70 mV.

Additional characterization is carried out using inside-out patches forsingle channel recordings. This method makes it simpler to studyendothelial cell patches, which can thus be obtained from either intactisolated capillaries or from single isolated endothelial cells. Inaddition, this method allows precise control of Ca²⁺, H⁺ and ATPconcentrations on the cytoplasmic side, and for this reason ispreferable to whole-cell recordings. Also, as previously shown (Chen etal., 2003), in this configuration anti-SUR1 antibody binds to thechannel and inhibits glibenclamide action, making positive,antibody-based identification of the channel readily feasible during thepatch clamp study.

The single channel slope conductance is obtained by measuring singlechannel currents at various membrane potentials using Na⁺, K⁺ and Cs⁺ asthe charge carrier, at different pH's including pH 7.9, 7.4, 6.9 and6.4.

The probability of channel opening (nP_(o)) is measured at differentconcentrations of intracellular calcium ([Ca²⁺]_(i)), at different pH'sincluding pH 7.9, 7.4, 6.9 and 6.4. The NC_(Ca-ATP) channel inastrocytes is regulated by [Ca²⁺]_(i), a unique feature thatdistinguishes the NC_(Ca-ATP) channel from K_(ATP) channel.

The concentration-response relationship is measured for channelinhibition by AMP, ADP, ATP at pH 7.9, 7.4, 6.9 and 6.4. There is apotentially important interaction between hydrogen ion and nucleotidebinding that may also be very important in the context of ischemia.

The concentration-response for channel inhibition by glibenclamide isstudied. The effect of glibenclamide will be studied at different pH's(7.9, 7.4, 6.9 and 6.4). The importance of these studies isseveral-fold. Pharmacological data at neutral pH are critical tocharacterizing the channel and for comparison with the channel inastrocytes. Values for half-maximum inhibition by sulfonylureas provideuseful information on involvement of SUR1 vs. other SUR isoforms andother potential targets. As discussed above, because glibenclamide andother sulfonylureas are weak acids, they are more lipid soluble at lowpH and thus can be expected to access the membrane more readily at lowpH. See detailed discussion and the effect of pH on channel inhibitionby glibenclamide in citation (Simard et al., 2008).

Statistical analysis. Means are compared using ANOVA.

In certain embodiments of the invention, SUR1 and TRPM4 areprogressively upregulated at both the protein and mRNA levels in theregion of percussion during the initial few hours post-injury, thatupregulation is prominent in neurons and capillary endothelial cells,and that upregulation requires several hours to reach a maximum.Moreover, in specific embodiments SUR1 and TRPM4 upregulation areassociated with formation of functional NC_(Ca-ATP) channels and thatKir6.x pore forming subunits are not involved.

Early treatment with the proper dose of the SUR1 antagonist,glibenclamide, minimizes formation of edema and progressive secondaryhemorrhage, and glibenclamide shifts the injury-magnitude vs. responsecurve to the right, in specific embodiments. There is data showing astrong salutary effect of glibenclamide when treatment is begunimmediately after percussion-TBI. The findings indicate that this drugis useful. Doses of drug and timing of drug administration is optimized.

The endpoints for study, edema and secondary hemorrhage, are reliablyquantified by measuring extravasated sodium and hemoglobin. The choiceof these measures reflects the embodiment that edema and secondaryhemorrhage are reliable, quantifiable indicators of lesion severity inthe acute phase, and correlate well with lesion size and neurobehavioralperformance assessed at later times, in certain cases.

Overview of Studies:

In a specific embodiment the effect of glibenclamide on edema andhemorrhage is determined when dosing and timing are varied. For thesestudies, rats re subjected to ˜3 atm percussion-TBI; 4 different timedelays (0-6 hr) before administration of one dose of drug (“dose2”, seebelow) are studied, and 4 different doses of drug when drug isadministered with a 2-hr delay are studied Each animal is evaluated foredema (sodium) and hemorrhage (hemoglobin) at 24 hr post-injury, atwhich time hemorrhage has maximized (see FIGS. 17A-17C).

Experiments useful to assess the effect and extent of glibenclamide onshifting the injury-magnitude vs. response curve for edema and forhemorrhage, separate groups of rats are studied that are injured withdifferent percussion pressures (˜1, ˜2, ˜3, ˜4 atm), and are treatedwith the “best dose” of glibenclamide, as determined in the foregoingstudies, with no delay in treatment.

Experimental Design:

Using edema (sodium) and hemorrhage (hemoglobin) as treatment endpoints,one can measure the effect of treatment with glibenclamide, starting atvarious times after injury (0-6 hr) and with various doses (4 differentdoses) of glibenclamide

One can study 11 groups of male rats with percussion-TBI, with 8rats/group, as follows, for example:

1. 0-hr delay/vehicle control 2. 0-hr delay/dose 2 3. 2-hr delay/vehiclecontrol 4. 2-hr delay/dose 2 5. 4-hr delay/vehicle control 6. 4-hrdelay/dose 2 7. 6-hr delay/vehicle control 8. 6-hr delay/dose 2 9. 2-hrdelay/dose 1 10. 2-hr delay/dose 3 11. 2-hr delay/dose 4

where:

dose1=loading dose, 2.5 μg/kg, i.p.; infusion rate, 75 ng/hr, s.q.

dose2=loading dose, 5 μg/kg, i.p.; infusion rate, 150 ng/hr, s.q.

dose3=loading dose, 10 μg/kg, i.p.; infusion rate, 300 ng/hr, s.q.

dose4=loading dose, 20 μg/kg, i.p.; infusion rate, 600 ng/hr, s.q.

vehicle control=DMSO (same amount as in dose2) in NS

These doses are calculated based on the following:

1. the volume of distribution for glibenclamide (in humans) is 0.2 L/kg.48

2. for the loading doses, the serum concentrations are 25, 50, 100, 200nM, based on the EC₅₀ value for channel inhibition (6 nM at pH 6.83).

3. lacking specific pharmacokinetic data for the rat, we base ourinfusion doses on our previous experience with stroke (Simard et al.,2006) and data with TBI (see above), which indicate that an infusionrate of 75-200 ng/hr are an effective rate. Overall, the data indicatethat 75 ng/hr, which has definite positive effects (Simard et al., 2006;Simard et al., 2008) is a suitable low dose, and that higher doses arealso suitable and may be preferred.

4. testing in uninjured rats as well as on rats with stroke and SCI todetermine the effect of these doses on serum glucose; of the dosessuggested above, only the highest are hypoglycemogenic, but only mildlyso. Notably, the loading doses of glibenclamide are 40-400 times lessthan typically used to induce hypoglycemia in rats (bd Elaziz et al.,1998).

Power analysis was performed with the following assumptions: α=0.05;tails=2; N=8/group; ratio for (raw difference between populationmeans)/(S.D. of one population)=2/1 (a conservative assumption, assuggested by FIGS. 17A-17C). These values yield a power of 96%likelihood of detecting a significant effect.

Specific Methods:

Delay of treatment: Mini-osmotic pumps are implanted within 2-3 min ofTBI. The pumps are fitted with widely-used “Lynch-coil” catheters thatprovide a dead space that requires the designated amount of time tofill. At the designated time, animals are also given the loading dose ofglibenclamide i.p.

Monitoring serum glucose: serum glucose is be monitored every 3-12 hrduring the first 24 hr after injury using a tail puncture to obtain adroplet of blood, and a standard glucometer for glucose measurements, toassure that levels are near euglycemic (80-160 mg/dL).

Preparation of tissues. After death, animals are perfused withheparinized PBS to remove intravascular blood. A 10-mm thick section ofthe upper half of the hemisphere encompassing the contusion isharvested.

Edema and hemorrhage: Tissue sodium and hemoglobin are measured insamples from the same homogenates. Sodium content is measured by flamephotometry, as described (Xi et al., 2001) Hemoglobin (Hgb) isquantified spectrophotometrically after conversion to cyanomethemoglobinusing Drabkin's reagent (Choudhri et al., 1997; Pfefferkorn andRosenberg, 2003). This method has been used by us for quantifyinghemorrhage following SCI in rats (Simard et al., 2007).

Data analysis: data obtained from vehicle-treated animals are comparedwith data obtained from glibenclamide-treated animals. Statisticalsignificance is assessed using ANOVA.

Using edema (sodium) and hemorrhage (hemoglobin) as treatment endpoints,the shift in the stimulus-response curve with the “best dose” ofglibenclamide administered without delay post-injury is measured, inseparate groups of rats injured with different impact pressures (˜1, ˜2,˜3, ˜4 atm)

These studies are similar to those above, except that the “best dose” ofglibenclamide (determined above) administered immediately after injuryis used. The choice of percussion pressures (˜1, ˜2, ˜3, ˜4 atm), isbased in part on the literature for fluid percussion (Thompson et al.,2005), and on experience with the magnitude of injury produced in amodel with 2.5-3 atm injury levels (see elsewhere herein).

Power analysis was performed with the following assumptions: α=0.05;tails=2; N=8/group; ratio for (raw difference between populationmeans)/(S.D. of one population)=2/1 (a conservative assumption, assuggested by FIGS. 17A-17C). These values yield a power of 96%likelihood of detecting a significant effect.

Specific methods: same as above

In specific embodiments, glibenclamide is beneficial in reducing edemaand hemorrhage in the area of percussion, at least for some doses andwith some delay in treatment, and shifts the injury-magnitude vs.response curve to the right, i.e., converts a “severe” injury to a“moderate” injury.

In certain embodiments, serum glucose levels are monitored to assurethat they do not drop too low (less than about 80 mg/dL). Inembodiments, the protocols are amended to correct for hypoglycemia, inorder to maintain levels between 80-160 mg/dL.

In certain embodiments, in a rodent model of TBI, treatment with the“best dose” of the sulfonylurea receptor antagonist, glibenclamide,improves early sensorimotor and later cognitive and psychophysiologicalperformance, and reduce lesion size and hippocampal neuronal cell loss.The foregoing studies are conducted with terminal endpoints (animalssacrificed to measure edema and blood in contused brain at 24 hr). Onecan perform measurements of neurofunctional, cognitive andpsychophysiological endpoints out to 6 months in separate groups of maleand female rats. These studies determine whether early treatment-relatedgains in edema and hemorrhage translate into long-term functional gains.In addition, these studies assess the role of gender in the response toglibenclamide treatment.

Animal and human studies have shown that the response to CNS injury isdifferent in females and males, and that gender affects behavioralperformance (Bimonte et al., 2000; Gresack and Frick, 2003; LaBuda etal., 2002). It is ascertained whether any difference in response toglibenclamide treatment exists between male and female rats, in certainaspects of the invention.

In humans post-TBI, the goals and targets of rehabilitation differ basedon time post-TBI. Early-on after injury, acute rehabilitation tends tofocus on recovery of sensorimotor dysfunction, locomotion, etc. Lateron, after sensorimotor abnormalities have stabilized, long termcognitive and psychophysiological effects become more important targetsof rehabilitation. One can assess the animals for effects of treatmentwith this time-frame in mind:

1. During the early phase, the following are assessed: (i) astrength/reflex test (NEUROLOGICAL SEVERITY SCORE); (ii) vestibulomotortests (ROTAROD TEST and SPONTANEOUS FORELIMB USE TASK).

2. Animals are then allowed to survive for 6 months, at which time onecan assess: (iii) a cognitive test (MORRIS WATER MAZE LEARNINGPARADIGM); (iv) fear conditioning test (SUSCEPTIBILITY TO STRESS-INDUCEDNONHABITUATING STARTLE).

This comprehensive range of testing includes sensorimotor tasks,cognitive and as well as a psychophysiological outcome measurepotentially related to delayed-onset PTSD, (Garrick et al., 2001; Cohenet al., 2004), a critical sequelae of TBI in humans (Andrews et al.,2007; Carty et al., 2006).

Overview of Experiments:

The animals undergo ˜3 atm percussion-TBI, are administered eithervehicle or drug, and later are assessed for neurofunctional andneurobehavioral recovery. One can use the “best dose” of glibenclamide,as determined in studies referred to above, and one can use twodifferent treatment times—treatment starting immediately post-injury andtreatment starting with a 4-hr delay, with both treatments lasting for 1week. However, an important purpose of the studies is to ascertainwhether a 4-hr delay in treatment is effective. In certain cases thestart of treatment is delayed in one group as long as possible afterinjury, in order to most usefully simulate the human situation.

Neurofunctional recovery is assessed using established sensorimotortests during post-injury days 1-28 (Fujimoto et al., 2004). Cognitiveand psychophysiological tests are assessed at 6 months. Body weight ismeasured periodically. Histological and stereological evaluation ofbrains, includes determining overall lesion size as well as neuronalcounts in CA(1)/CA(3) hippocampal regions at 6 months.” (Grady et al.,2003; Hellmich et al., 2005).

(A) NEUROLOGICAL SEVERITY SCORE (NSS). This is an aggregate neurologicaltesting strategy (Fujimoto et al., 2004). In the Neurologic SeverityScore (see Table 5 of Fujimoto et al., 2004), animals are scored on anall-or-none scale for such tests as the ability to exit from a circle,righting reflex, hemiplegia, limb reflexes, pinna reflex, cornealreflex, startle reflex, beam balance, and beam walking. An animalreceives one point for the ability to successfully perform each task andno points for the inability to perform, with the overall NSS being thesum of these scores.

(B) ROTAROD TEST. (Hamm et al., 1994; Lu et al., 2003) The rotarod taskis a sensitive index of injury-induced motor dysfunction. The rotarodtask measures aspects of motor impairment that are not assessed byeither the beam-balance or beam-walking latency, and has been found tobe a more sensitive and efficient index for assessing motor impairmentproduced by brain injury. (Hamm et al., 1994) Frequency of evaluationcan affect performance—daily assessment promotes functional recoverywhereas weekly assessment does not significantly affect outcome ininjured animals during a 4-week assessment. (O'Connor et al., 2003).

(C) SPONTANEOUS FORELIMB USE TASK (SFU). This task measures sensorimotorasymmetry. (Schallert et al., 2000) It involves placing the animal in aplastic cylinder and determining the amount of time the animal spendsrearing with the left, right, or both forelimbs on the cylinder wall.The cylindrical shape encourages vertical exploration of the walls withthe forelimbs and it allows evaluation of landing activity. This testhas been shown to be effective in detecting an injury deficit up to fivemonths after controlled cortical impact in a mouse model. (Baskin etal., 2003). In addition, quantification of time spent in verticalexploration gives an overall measure of spontaneous activity.

(D) MORRIS WATER MAZE LEARNING PARADIGM (MWM) (Thompson et al., 2006;Dixon et al., 1999; Sanders et al., 1999; Kline et al., 2002). The MWMis the most widely used test for cognitive evaluation in experimentalTBI. (Fujimoto et al., 2004). Deficits in learning have been detected upto 1 year post-injury in rats. (Fujimoto et al., 2004).

(E) STRESS-INDUCED NONHABITUATING STARTLE. The interest in the startleresponse is two-fold. First, it is known that percussion-TBI in ratsyields a depressed startle response that can persist for over 30 days(Dixon et al., 1987; Lu et al., 2003; Wiley et al., 1996) possiblyreflecting the overall decrease in spontaneous activity post-TBI. Thus,in its simplest form, the startle response provides a good test of theeffect of glibenclamide treatment, with treatment expected to normalizeor partially normalize this response. Note that the simple startleresponse in part of the NSS, is assessed during the early recovery phase(first 28 days).

It is believed that TBI-induced limbic system damage observed inpercussion models of TBI may predispose the animal to delayedpsychophysiological abnormalities. Months after injury, maladaptive“rewiring” of limbic circuitry is believed to give rise to alteredpsychophysiological responses, e.g., an increase in the susceptibilityto non-habituating startle induced by new, consciously-experiencedstress. A link between injury to limbic structures with increasedsusceptibility to non-habituating or augmented sensorimotor responses,has been discussed by Harvey et al., 2003, and is based on theobservation of the important role of the hippocampus in the extinctionof conditioned fear. (Brewin, 2001). Thus, whereas early-on, TBI isbelieved to be associated with depressed startle responses, later“recovery” from TBI is surprisingly believed to be lower the thresholdfor the “intensity” of a new stress (strength, duration or number ofrepetitions) that is required to induce non-habituating startle.

The interest in non-habituating startle resides in its potentialrelevance to post-traumatic stress disorder (PTSD). In humans followingexposure to trauma, a vulnerable sub-population of individuals developsPTSD with characteristic persistent autonomic hyper-responsivity,increased sensory arousal, increased startle response, and alteredhypothalamo-pituitary-adrenal regulation. Often, onset of these symptomsis delayed. (Andrews et al., 2007; Carty et al., 2006). Similar effectsare seen in (uninjured) rats in a rodent models of PTSD, in which the(awake) animal is exposed to repeated, randomly applied, inescapablestress. The stress paradigm used by Manion et al. (2007) consisted of2-hr sessions of immobilization and randomly applied tailshocks each dayfor 3 days. Seven days later, the rats developed non-habituatingstartle. Slightly different paradigms have been used by others (Garricket al., 2001; Garrick et al., 1997; Rasmussen et al., 2008). The methodsdisclosed herein may be used to evaluate the effect of glibenclamide onthis phenomenon post-TBI. one can assess this question, and evaluate theeffect of glibenclamide on this phenomenon post-TBI.

Experimental Design:

The effect of the “best dose” of glibenclamide administered at twotreatment times on neurofunctional, cognitive and psychophysiologicalrecovery is assessed in animals in times extending out to 6 months afterinjury.

8 groups are studied in all, 4 groups of males and 4 groups of females;for each gender, there is one sham-injured group and three TBI groups;the three TBI groups include a vehicle-treated group, a group treatedwith the “best dose” glibenclamide given immediately after injury, and agroup treated with the “best dose” glibenclamide given 4 hr afterinjury. The “best dose” is determined from studies described above.

On any given day, 2 rats undergo TBI and then enter into a schedule ofcomprehensive testing during the subsequent 4 weeks (followed by 5 monthrecovery and more testing). Gender and treatment group are randomlyassigned.

Power analysis was performed with the following assumptions: α=0.05;tails=2; N=12/group; ratio for (raw difference between populationmeans)/(S.D. of one population)=3/2 (worse case scenario). These valuesyield a power of 94% likelihood of detecting a significant effect.

Specific Methods:

Neurological severity score (NSS). The Neurologic Severity Score isobtained as detailed in Table 5 of Fujimoto et al. (2004).

FREQUENCY OF TESTING POST-TBI: Rats are tested on days 1, 3, 7, 14, 21,28 post-TBI.

STATISTICAL TEST: Repeated measures ANOVA.

Rotarod test. The accelerating Rotarod test has been described. Rats aretrained for 3 consecutive days before TBI, measuring latency to fall offthe rod (10 trials/day).

FREQUENCY OF TESTING POST-TBI: Rats are tested on days 3, 7, 14, 21, 28post-TBI. This schedule avoids the potential confounder that frequentassessments tend to promote functional recovery whereas weeklyassessments do not (O'Connor et al., 2003).

STATISTICAL TEST: Repeated measures ANOVA.

Spontaneous forelimb use task (SFU). Rats are placed in a clear cylinder(diameter, 20 cm; height, 20 cm) in front of a mirror. Activity isvideotaped for 5-30 min, depending on activity levels. Scoring is doneby an experimenter blind to the condition of the animal using a VCR withslow motion and frame by frame capabilities. Asymmetrical forelimb usageis counted. This consists of recording: (1) the limb (left or right)used to push off the floor prior to rearing; (2) the limb used forsingle forelimb support on the floor of the box; and (3) the limb usedfor single forelimb support against the walls of the box (Schallert etal., 2000). Usage of both forelimbs simultaneously is not counted. Dataare expressed as percentage of right (unaffected by injury) forelimbuse, i.e. (right forelimb use/right+left forelimb use)

0100.

FREQUENCY OF TESTING POST-TBI: Rats are tested on days 3, 7, 14, 21, 28post-TBI, during the same session with Rotarod.

STATISTICAL TEST: Repeated measures ANOVA.

Morris water maze learning paradigm (MWM). The MWM will be used tomeasure acquisition of spatial learning (DeFord et al., 2001; Hamm etal., 1993). A standard apparatus is used. At each trial, rats are placedby hand in the pool at one of four start locations (north, south, east,west) facing the wall. Start locations are randomly assigned to eachanimal. A computerized video tracking system is used to record theanimal's latency to reach the goal. The tracking program calculates thedistance from the animal to the goal during each trial (at 0.2 secintervals) and adds these distances together as a measure of how closethe animal is swimming to the goal during the trial. This measure isdefined as “cumulative distance from the goal.” To assess for thepossible confounding effect of motor impairment, swim speeds are alsomeasured on each trial. Rats are given a maximum of 120 sec to find thehidden platform. If an animal fails to find the platform after 120 sec,it is placed on the platform by the experimenter. Rats are allowed toremain on the platform for 30 sec and then are returned to a cage with alamp warmer between trials. There is a 4-min inter-trial interval.Animals are tested 6 months post-TBI to allow for recovery of motordeficits. Rats were given four trials per day for five consecutive days.

FREQUENCY OF TESTING POST-TBI: 4 trials/day on 5 consecutive days,beginning 6 months post-TBI).

STATISTICAL TEST: Repeated measures ANOVA.

Stress-induced non-habituating startle (SINHS) (Manion et al., 2007).Animals are acclimated to the acoustic startle equipment for 3consecutive days, one day without sound followed by two days with sound.This acclimation is finished 3 days prior to baseline recordings inorder to avoid desensitization effects. A baseline recording of acousticstartle response (details below) is taken for each animal on the dayprior to beginning the stress procedure. Stress exposure consists of a2-h per day session of immobilization and tail-shocks for threeconsecutive days. Stressing is done during the dark or active phase ofthe light-dark cycle. Animals are restrained by being wrapped in a clothjacket and having their head and torso immobilized in a ventilatedplexiglass tube. Forty electric shocks (2-3 mA, 3 s duration;programmable animal shocker, Coulbourn Instruments) are delivered totheir tails at semi-random intervals of 150-210 s.

ASR testing is conducted with a Startle Response Acoustic Test System(San Diego Instruments). This system includes weight-sensitiveplatform(s) in a sound-attenuated chamber. The animal's movements inresponse to stimuli are measured as a voltage change by a strain gaugeinside each platform and are converted to grams of body weight changefollowing analog to digital conversion. These changes are recorded by aninterfaced computer as the maximum response occurring within 200 ms ofthe onset of the startle-eliciting stimulus. All acoustic stimuli areadministered by an amplified speaker mounted 24 cm above the test cage.During testing, animals are individually placed in holding cages(14.5×7×6.5 cm) that are small enough to restrict extensive locomotionbut large enough to allow the subject to turn around and make othersmall movements.

Following placement of the animal into the chamber, the chamber lid isclosed, leaving the subject in darkness. A 3 min adaptation periodoccurs in which no startle stimulus is presented. Startle stimuliconsist of 110 dB sound pressure level (unweighted scale; re: 0.0002dynes/cm2) noise bursts of 20 ms duration, sometimes preceded by 100 mswith 68 dB, 1 kHz pure tones (pre-pulses). Decibel levels are verifiedby a sound meter. Each stimulus had a 2 ms rise and decay time such thatonset and offset are abrupt, a primary criterion for startle. There arefour types of stimulus trials: 110 dB alone, with pre-pulse, pre-pulsealone and no stimulus. Each trial type is presented eight times. Trialtypes are presented in random order to avoid order effects andhabituation. Inter-trial intervals range randomly from 15 to 25 s. Allanimals are tested 1, 4, 7 and 10 days following the final day of thestress procedure, which will begin 1 week after the MWM, 6 monthspost-TBI.

FREQUENCY OF TESTING POST-TBI: 1 trial/day on 13 consecutive days,starting 1 week after MWM, 6 months post-TBI.

STATISTICAL TEST: Repeated measures ANOVA.

The effect of the “best dose” of glibenclamide administered at twotreatment times on lesion size and hippocampal neuronal count at 6months post-injury is assessed.

These experiments utilize the brains of animals injured and treated inthe earlier portion of this example, using tissues from 5 rats from eachof the 8 treatment groups. Coronal sections (25 μm) spaced 200 μm apartthroughout the injury area (5 mm rostral and 5 mm caudal to theepicentre) are stained with Niss1 stain and adjacent sections areimmunolabeled for NeuN (Chemicon).

A stereological system is used for efficient, unbiased and accuratemeasurements of lesion volumes and of counts of surviving neurons indifferent treatment groups. Niss1 stained sections are used to measurelesion size. NeuN-immunolabeled sections are used to count neurons inipsilateral and contralateral hippocampus (CA1, CA3 and dentate gyrus).All quantitative analyses are performed blindly. Using theStereoinvestigator software (Microbrightfield, Williston, Vt., USA),counts of neurons (450×450 μm grids) and neuronal profiles within 50×50μm counting frames spaced evenly throughout the ipsilateral andcontralateral hippocampus are obtained using a 20× objective. UsingStereoinvestigator software, serial reconstruction of the ipsilateraland contralateral hippocampus are performed to compute total volumes. Todetermine if the neurons are decreasing in size, cross-sectional areasof hippocampal neuronal profiles will be determined by outlining theperimeter of all defined neurons within 50×50 μm counting frames spacedevenly throughout the sections (450×450 μm grids).

[Statistical Test: ANOVA.

In particular embodiments, glibenclamide, as an example, results in asignificant improvement in standard measures neurofunctional outcome,including the neurological severity score and vestibulomotorassessments, and the beneficial effects endure during the month ofrepeated testing.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

Patents and Patent Applications

U.S. Pat. No. 5,399,363

U.S. Pat. No. 5,466,468

U.S. Pat. No. 5,543,158

U.S. Pat. No. 5,580,579

U.S. Pat. No. 5,629,001

U.S. Pat. No. 5,641,515

U.S. Pat. No. 5,725,871

U.S. Pat. No. 5,756,353

U.S. Pat. No. 5,780,045

U.S. Pat. No. 5,792,451

U.S. Pat. No. 5,804,212

U.S. Pat. No. 6,613,308

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1.-48. (canceled)
 49. A method of targeting a plasma level of glyburidein an individual, comprising the step of administering glyburide to theindividual as follows: (a) an intravenous loading dose of glyburide; and(b) a maintenance dose of glyburide, which is different than the loadingdose.
 50. The method of claim 49, wherein the loading dose of glyburideis 30-90 times the amount of the maintenance dose.
 51. The method ofclaim 50, wherein the loading dose of glyburide is 40-80 times theamount of the maintenance dose.
 52. The method of claim 50, wherein theloading dose of glyburide is 50-60 times the amount of the maintenancedose.
 53. The method of claim 49, wherein the loading dose is 15.7 ug to665 ug.
 54. The method of claim 53, wherein the loading dose is 15.7 ugto 117 ug.
 55. The method of claim 49, wherein the loading dose is 117ug to 665 ug.
 56. The method of claim 49, wherein the maintenance doseis 0.3 ug/min to 11.8 ug/min.
 57. The method of claim 56, wherein themaintenance dose is 0.3 ug/min to 2.1 ug/min.
 58. The method of claim49, wherein the total daily dose is 432 ug/day to 17 mg/day.
 59. Themethod of claim 58, wherein the total daily dose is 432 ug/day to 3mg/day.
 60. The method of claim 49, wherein the targeted plasma level ofglyburide is from 4.07 ng/ml to 160.2 ng/ml.
 61. The method of claim 60,wherein the targeted plasma level of glyburide is from 4.07 ng/ml to28.3 ng/ml.
 62. The method of claim 49, wherein the glyburide isadministered to said individual for 3 days to 7 days.
 63. The method ofclaim 62, wherein the glyburide is administered to said individual for 3days to 5 days.